INFLUENCE OF STORAGE DURATION, HARVESTING STAGE AND CALCIUM TREATMENT ON THE STORAGE PERFORMANCE OF

BY

IBADULLAH JAN

Dissertation submitted to Khyber Pakhtunkhwa Agricultural University Peshawar in partial fulfillment of the requirement for the Degree of

DOCTOR OF PHILOSOPHY IN AGRICULTURE (HORTICULTURE)

DEPARTMENT OF HORTICULTURE FACULTY OF CROP PRODUCTION SCIENCES KHYBER PAKHTUNKHWA AGRICULTURAL UNIVERSITY PESHAWAR -PAKISTAN JULY, 2011

TABLE OF CONTENTS

Chapter No. Title Page No.

ABSTRACT i

ACKNOWLEDGEMENTS iii

LIST OF TABLES iv

LIST OF FIGURES v

LIST OF APPENDICES ix

CHAPTER No. 1

GENERAL INTRODUCTION 1

CHAPTER No. 2

REVIEW OF LITERATURE 6

CHAPTER No. 3

INFLUENCE OF STORAGE DURATION ON OF PHYSICO-

CHEMICAL CHANGES IN FRUIT OF APPLE CULTIVARS

1. ABSTRACT 24

2. INTRODUCTION 25 3. MATERIALS AND METHODS 28 4. RESULTS 34 5. DISCUSSIONS 45 6. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS 52 CHAPTER No. 4 STORAGE PERFORMANCE OF APPLE CULTIVARS HARVESTED AT DIFFERENT STAGES OF MATURITY 1. ABSTRACT 54 2. INTRODUCTION 55 3. MATERIALS AND METHODS 57

4. RESULTS 60 5. DISCUSSIONS 77 6. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS 85

CHAPTER No. 5

INFLUENCE OF CaCl2 CONCENTRATION AND DIPPING

DURATION ON PHYSICO CHEMICAL CHANGES IN APPLE cv. ‘

1. ABSTRACT 87 2. INTRODUCTION 88 3. MATERIALS AND METHODS 90 4. RESULTS 91 5. DISCUSSIONS 116 6. SUMMARY,CONCLUSIONS AND RECOMMENDATIONS 123 CHAPTER No. 6

INFLUENCE OF CaCl2 TREATMENT ON STORAGE

PERFORMANCE OF APPLE CULTIVARS

1. ABSTRACT 125

2. INTRODUCTION 126 3. MATERIALS AND METHODS 127 4. RESULTS 129 5. DISCUSSIONS 149 6. SUMMARY,CONCLUSIONS AND RECOMMENDATIONS 156

OVERALL CONCLUSIONS AND RECOMMENDATIONS 158

LITERATURE CITED 159

APPENDICES 177

INFLUENCE OF STORAGE DURATION, HARVESTING STAGE AND CALCIUM TREATMENT ON THE STORAGE PERFORMANCE OF APPLE CULTIVARS

Ibadullah Jan and Abdur Rab Department of Horticulture, Khyber Pakhtunkhwa Agricultural University, Peshawar

ABSTRACT The “Influence of storage duration, harvesting stage and calcium treatment on the storage performance of apple cultivars” was conducted at Department of Horticulture, Khyber Pakhtunkhwa Agricultural University Peshawar, Pakistan during the years 2007-10 to optimize the storage duration and calcium treatments for apple fruit. The fruit of apple (Pyrus domestica L.) cultivars: Royal , Mondial Gala, and Red Delicious were harvested at optimum maturity and stored at 5±1°C with 60-70% relative humidity. Physico-chemical changes in fruit were determined at 30 days interval during storage. Significant differences were observed among apple cultivars. Red Delicious had the highest juice content (58.47%), TSS/Acid ratio (23.12), ascorbic acid (13.12 mg/100g), fruit firmness (5.98 kg/cm2), fruit density (0.82 g/cm3) as well as the least weight loss (2.22%) but also had the highest bitter pit (11.86%) and soft rot (13.53%) incidence. Titratable acidity was the highest (0.55%) in cultivar Mondial Gala and starch score was the maximum (5.22) in cultivar Golden Delicious. Storage resulted in significant increased in weight loss, total soluble solids, total sugar, pH, TSS/Acid ratio, bitter pit incidence and soft rot, while juice content, starch score, titratable acidity, ascorbic acid, firmness and density of fruit decreased with increasing storage duration. Physico-chemical characteristics of apple fruit varied significantly with harvesting stage and storage duration. The juice content (47.68%), total soluble solids (10.07), total sugar (9.31%), pH (3.71), TSS/Acid ratio (18.73), ascorbic acid (10.11 mg/100g) and soft rot (9.52%) recorded with early mature fruit, increased to 59.33%, 12.92, 12.98%, 4.23, 29.29, 12.50% and 15.22% accordingly in late mature fruits, while weight loss (3.34%), starch score (4.95), titratable acidity (0.59%), fruit firmness (5.88 kg/cm2), density of fruit (0.82 g/cm3) and bitter pit incidence (11.69%) recorded

at early maturity stage, declined with delaying the harvesting to 1.93%, 3.21, 0.49%, 4.81 kg/cm2, 4.81 g/cm3 and 6.63% respectively at late maturity stage. Dipping in calcium solution declined the storage related changes but the influence was dependant on concentration as well as dipping duration. The juice content (60.63%), starch score (5.05), ascorbic acid (12.67 mg/100g), firmness (5.98 kg/cm2) and density of fruit (0.81 mg/100g) recorded in fruits dipped in 0% CaCl2 solution (control), increased to 64.34%, 5.68, 13.87 mg/100g, 6.54 kg/cm2 and 0.84 mg/100g accordingly in fruits dipped in 9% CaCl2 solution. By contrast, weight loss (1.95%), total soluble solids (12.01), total sugar (10.95%), TSS/Acid ratio (28.09), bitter pit incidence (15.18%) and soft rot (15.33%) incidence recorded in control, decreased to

1.31%, 11.88, 10.76%, 23.88, 3.80% and 2.10% in fruits treated with 9% CaCl2 solution. The starch score (5.30), firmness (6.13 kg/cm2) and density of fruit (0.82 mg/100g) recorded in fruits dipped for 3 minutes in CaCl2 solution, increased to 5.50, 2 6.49 kg/cm and 0.84 mg/100g accordingly in fruits dipped for 12 minutes in CaCl2 solution, whereas weight loss (1.81%), TSS/Acid ratio (27.21), bitter pit incidence (11.92%) and soft rot (7.07%) incidence recorded in fruits dipped for 3 minutes in

CaCl2 solution, reduced to 1.41%, 25.27, 5.60% and 6.28% respectively in fruits treated CaCl2 solution for 12 minutes. It can be concluded that cultivar Red Delicious had superior quality but more susceptible bitter pit and soft rot with prolong storage. Harvesting at mid mature stage and calcium chloride (9%) treatment for 9 minutes resulted in enhanced storage performance.

ACKNOWLEDGEMENT I have no words to express my deepest sense of gratitude to Almighty Allah, the Most Merciful, the Beneficent, Who bestowed upon me the courage and will to complete this project, and contribute to the noble field of knowledge. Cordial gratitude to the Prophet Muhammad (P.B.U.H) who is forever a torch of guidance and knowledge for humanity. I wish to express my deepest gratitude and profound regard to my honorable supervisor Prof. Dr. Abdur Rab, Department of Horticulture for his constant encouragement, helpful suggestions and guidance during my scholastic life. His critical insight, consistent advice, constructive criticism, personal interest and supervision, generated the vigor for excellence in its pursuits, without which it would not have been possible to undertake this research project. I feel pleasure to thank members of my supervisory committee, Prof. Dr. Amanullah Jan, Department of Agronomy, for their cooperation excellent supervision, guidance and valuable suggestions. I am also thankful to Prof. Dr. Noor-Ul-Amin, Chairman Department of Horticulture, Prof. Dr. Farhatullah, Director Advance Studies and Research (DASAR), Prof. Dr. Zahoor Ahmad Swati, Dean Faculty of Crop Production Sciences for their cooperation. I am highly indebted to Dr. Muhammad Arif (Asstt. Prof), Department of Agronomy for his help in data analysis. I also extend many thanks to Prof. Dr. Nawab Ali, Prof. Dr. Sher Muhammad, Dr. Muhammad Zubair, Dr. Abdul Mateen and Dr. Abrar Hussain Shah, Dr. Gohar Ayub and Mr. Mehboob Alam, Department of Horticulture for their full support during my research work. Thanks are also extended to Dr. Muhammad Sajid, Assistant Professor and Syed Tanveer Shah, Lecturer, Department of Horticulture for providing sincere help and cooperation. I gratefully acknowledge Higher Education Commission, Islamabad for providing full financial during my studies. Finally, I would feel incomplete without thanking to my parents, wife, brothers, sisters and other family members who tolerated me patiently during this critical period and their prayers enabled me to complete my Ph.D work successfully.

IBADULLAH JAN

LIST OF TABLES

Table No. Description Page No.

Table 3.1 The effect of storage duration on weight loss (%), percent 36 juice (%), starch, TSS (%) and total sugar (%) of apple cultivars

Table 3.2 The effect of storage duration on percent titratable acidity, pH, 39 TSS/Acid ratio and ascorbic acid (mg/100g) of apple cultivars

Table 3.3 The effect of storage duration on firmness (kg/cm2), density 43 of fruit (g/cm3), soft rot (%) and bitter pit (%) of apple cultivars

Table 4.1. Calcium content (mg/kg) of orchard soil at the time of fruit 58 picking

Table 4.2. Calcium content (%) of apple leaves at the time of fruit picking 58

Table 4.3 The effect of harvesting stages and storage on weight lost (%), 63 percent juice, starch, TSS (%) and total sugar (%) of apple cultivars

Table 4.4 The effect of harvesting stages and storage on percent titratable 69 acidity, pH, TSS/Acid ratio and ascorbic acid (mg/100g) of apple cultivars

Table 4.5 The effect of harvesting stages and storage on firmness (kg/cm2), 74 fruit density (g/cm3), soft rot (%) and bitter pit (%) of apple cultivars

Table 5.1 The influence of storage, dipping duration and CaCl2 conc- 96 entration on weight lost (%), percent juice, starch, TSS (%) and total sugar (%) of apple cultivars

Table 5.2 The influence of storage, dipping duration and CaCl2 concen- 109 tration on ascorbic acid (mg/100g), firmness (kg/cm2), fruit density (g/cm3), bitter pit (%) and soft rot (%) of apple cultivars

Table 5.3 Calcium content (mg/kg) of apple fruit cv Red delicious as affected 115 by CaCl2 concentration and dipping duration

Table 6.1 The effect of cultivars, storage and CaCl2 application on 133 weight lost, percent juice, starch, Titratable acidity (%), Total sugar (%), TSS (%) and TSS/Acid ratio of apple cultivars

Table 6.2 The effect of calcium application and storage on ascorbic acid 142 (mg/100g), firmness (kg/cm2), fruit density (g/cm3), soft rot (%) and bitter pit (%) of apple cultivars

Table 6.3 Effect of CaCl2 dipping on calcium content (mg/kg) of apple cultivars 148 LIST OF FIGURES

Figure No. Description Page No.

Figure 3.1 Interaction effect of storage duration and cultivar on asco- 40 rbic acid (mg/100g) of apple fruit

Figure 3.2 Interaction effect of storage duration and cultivar on bitter pit 44 incidence (%) of apple fruit

Figure 3.3 Interaction effect of storage duration and cultivar on soft rot (%) 44 of apple fruit

Figure 4.1 Variation in percent weight loss among apple cultivars after 64 150 days storage

Figure 4.2 Interaction effect of cultivar and harvesting stage on percent 64 weight loss in apple fruit

Figure 4.3 Influence of harvesting stages on percent weight loss in apple 65 fruits after 150 days storage

Figure 4.4 Interaction effect of cultivar and harvesting stage on percent 65 weight loss of apple fruits stored for 150 days

Figure 4.5 Interaction effect of storage and harvesting stage on total soluble 66 solids of apple

Figure 4.6 Interaction effect of storage and harvesting stage on total 66 sugar of apple

Figure 4.7 Interaction effect of storage and harvesting stage on titratable 70 acidity (%) of apple

Figure 4.8 Interaction effect of cultivar and storage on pH of apple fruits 70

Figure 4.9 Interaction effect of cultivar and storage ascorbic acid 71 (mg/100g) of apple

Figure 4.10 Interaction effect of storage and harvesting stage on ascorbic 71 acid (mg/100g) of apple

Figure 4.11 Interaction effect of cultivar and harvesting stage on bitter 75 pit (%) of apple

Figure 4.12 Influence of harvesting stages on bitter pit (%) of apple fruits 75 after 150 days storage

Figure 4.13 Interaction effect of cultivar and harvesting stage on soft 76 rot (%) of apple

Figure 4.14 Influence of harvesting stages on soft rot (%) of apple fruits 76 stored for 150 days

Figure 5.1 Influence of CaCl2 concentration on percent weight loss in apple 97 fruits stored for 150 days

Figure 5.2 Influence of dipping duration on percent weight loss in apple fruits 97 stored for 150 days

Figure 5.3 Interaction effect of CaCl2 concentration and dipping 98 duration on percent weight loss in apple fruit

Figure 5.4 Interaction effect of CaCl2 concentrations and storage on 98 percent juice of apple fruit

Figure 5.5 Interaction effect of CaCl2 concentration and storage on starch 99 score of apple

Figure 5.6 Interaction effect of dipping duration and storage on starch 99 scores of apple

Figure 5.7 Interaction effect of CaCl2 concentration and dipping duration 100 on starch scores of apple

Figure 5.8 Interaction effect of CaCl2 concentration and storage duration 100 on TSS of apple

Figure 5.9 Interaction effect of dipping duration and storage duration on TSS 101 of apple

Figure 5.10 Interaction effect of CaCl2 concentration and storage duration 101 on total sugar (%) of apple

Figure 5.11 Interaction effect of CaCl2 concentration and storage 102 duration on titratable acidity (%) of apple

Figure 5.12 Interaction effect of dipping duration and storage duration on 102 titratable acidity (%) of apple

Figure 5.13 Interaction effect of CaCl2 concentration and storage duration 103 on TSS/Acid ratio of apple

Figure 5.14 Interaction effect of dipping duration and storage duration 103 on TSS/Acid ratio of apple

Figure 5.15 Interaction effect of CaCl2 concentrations, dipping durations 104 and storage durations on TSS/Acid ratio of apple

Figure 5.16 Interaction effect of CaCl2 concentration and storage duration 110 on ascorbic acid (mg/100g) of apple

Figure 5.17 Interaction effect of CaCl2 concentration and storage duration 110 on fruit flesh firmness (kg/cm2) of apple

Figure 5.18 Interaction effect of dipping duration and storage duration on 111 fruit flesh firmness (kg/cm2) of apple

Figure 5.19 Interaction effect of CaCl2 concentration and storage duration on 111 fruit density (g/cm3) of apple

Figure 5.20 Influence of CaCl2 concentrations on bitter pit (%) of apple fruits 112 after 150 days storage

Figure 5.21 Influence of dipping durations on bitter pit (%) of apple fruits 112 stored for 150 days

Figure 5.22 Interaction effect of CaCl2 concentration and dipping duration on 113 bitter pit (%) of apple

Figure 5.23 Influence of CaCl2 concentration and dipping duration on bitter 113 pit (%) of apple fruits stored for 150 days

Figure 5.24 Effect of CaCl2 concentration on soft rot (%) incidence in apple 114 fruits stored for 150 days

Figure 5.25 Influence of dipping duration on soft rot (%) incidence in apple 114 fruits after 150 days storage

Figure 6.1 Variation in weight loss (%) in apple fruits stored for 150 days 134 of different apple cultivars

Figure 6.2 Effect of CaCl2 concentrations on weight loss (%) in apple fruits 134 stored for 150 days

Figure 6.3 Interaction effect of CaCl2 application and storage duration on 135 starch scores of apple

Figure 6.4 Interaction effect of CaCl2 application and storage duration on 135 TSS of apple

Figure 6.5 Interaction effect of CaCl2 application and storage duration on 136 total sugar (%) of apple

Figure 6.6 Interaction effect of cultivar and storage duration on titratable 136 acidity (%) of apple Figure 6.7 Interaction effect of CaCl2 application and storage duration 137 on titratable acidity (%) of apple

Figure 6.8 Interaction effect of CaCl2 application and storage duration 137 on TSS/Acid ratio of apple

Figure 6.9 Interaction effect of cultivar and storage on ascorbic acid 143 (mg/100g) of apple

Figure 6.10 Interaction effect of CaCl2 application and storage duration on 143 ascorbic acid (mg/100g) of apple

Figure 6.11 Interaction effect of cultivar and storage duration on fruit 144 flesh firmness (kg/cm2) of apple fruit

Figure 6.12 Interaction effect of CaCl2 application and storage duration on 144 fruit flesh firmness (kg/cm2) of apple

Figure 6.13 Variation in bitter pit (%) incidence in apple fruits after 150 145 days storage of different cultivars

Figure 6.14 Interaction effect of cultivar and storage on bitter pit (%) incidence 145 in apple

Figure 6.15 Effect of CaCl2 concentrations on bitter pit (%) incidence in 146 apple fruits after 150 days storage

Figure 6.16 Interaction effect of cultivar and storage on bitter pit (%) 146 of apple cultivar stored for 150 days

Figure 6.17 Influence of 150 days storage on soft rot (%) incidence in 147 apple cultivars

Figure 6.18 Effect of CaCl2 concentrations on soft rot (%) incidence in apple 147 fruits stored for 150 days

LIST OF APPENDICES

Appendix No. Description Page No.

1. ANOVA for weight loss (%) of apple cultivars during storage 177

2. ANOVA for juice content (%) of apple cultivars during storage 177

3. ANOVA for starch (score) of apple cultivars during storage 177

4. ANOVA for total soluble solids of apple cultivars during storage 177

5. ANOVA for total sugar (%) of apple cultivars during storage 178

6. ANOVA for percent acidity of apple cultivars during storage 178

7. ANOVA for pH (%) of apple cultivars during storage 178

8. ANOVA for TSS/Acid ratio of apple cultivars during storage 178

9. ANOVA for ascorbic acid (mg/100g) of apple cultivars during 179 storage

10. ANOVA for firmness (kg/cm2) of apple cultivars during storage 179

11. ANOVA for fruit density (g/cm3) of apple cultivars during storage 179

12. ANOVA for bitter pit (%) of apple cultivars during storage 179

13. ANOVA for percent soft rot of apple cultivars during storage 180

14. ANOVA for weight loss (%) of apple cultivars as affected by 180 storage and harvesting stages

15. ANOVA for juice content (%) of apple cultivars as affected by 180 storage and harvesting stages

16. ANOVA for starch (score) of apple cultivars as affected by 181 storage and harvesting stages

17. ANOVA for percent acidity of apple cultivars as affected by 181 storage and harvesting stages

18. ANOVA for total soluble solids of apple cultivars as affected 181 by storage and harvesting stages

19. ANOVA for total sugar (%) of apple cultivars as affected by 182 storage and harvesting stages

20. ANOVA for pH (%) of apple cultivars as affected by storage 182

and harvesting stages

21. ANOVA for TSS/Acid ratio of apple cultivars as affected by 182 storage and harvesting stages

22. ANOVA for ascorbic acid (mg/100g) of apple cultivars as affected 183 by storage and harvesting stages

23. ANOVA for firmness (kg/cm2) of apple cultivars as affected by 183 storage and harvesting stages

24. ANOVA for fruit density (g/cm3) of apple cultivars as affected by 183 storage and harvesting stages

25. ANOVA for bitter pit (%) of apple cultivars as affected by storage 184 and harvesting stages

26. ANOVA for percent soft rot of apple cultivars as affected by 184 storage and harvesting stages

27. ANOVA for weight loss (%) of apple fruit as affected by 184 storage, CaCl2 concentrations and dipping durations

28. ANOVA for juice content (%) of apple fruit as affected by 185 storage, CaCl2 concentrations and dipping durations

29. ANOVA for starch (score) of apple fruit as affected by storage, 185 CaCl2 concentrations and dipping durations

30. ANOVA for percent acidity of apple fruit as affected by 185 storage, CaCl2 concentrations and dipping durations

31. ANOVA for total soluble solids of apple fruit as affected by 186 storage, CaCl2 concentrations and dipping durations

32. ANOVA for total sugar (%) of apple fruit as affected by storage, 186 CaCl2 concentrations and dipping durations

33. ANOVA for TSS/Acid ratio of apple fruit as affected by storage, 186 CaCl2 concentrations and dipping durations

34. ANOVA for ascorbic acid (mg/100g) of apple fruit as affected by 187 storage, CaCl2 concentrations and dipping durations

35. ANOVA for firmness (kg/cm2) of apple fruit as affected by 187 storage, CaCl2 concentrations and dipping durations

36. ANOVA for fruit density (g/cm3) of apple fruit as affected by 187 storage, CaCl2 concentrations and dipping durations

37. ANOVA for bitter pit (%) of apple fruit as affected by storage, 188 CaCl2 concentrations and dipping durations

38. ANOVA for percent soft rot of apple fruit as affected by storage, 188 CaCl2 concentrations and dipping durations

39. ANOVA for weight loss (%) of apple cultivars as affected by 188 storage and CaCl2

40. ANOVA for juice content (%) of apple cultivars as affected by 189 storage and CaCl2

41. ANOVA for starch (score) of apple cultivars as affected by 189 storage and CaCl2

42. ANOVA for percent acidity of apple cultivars as affected by 189 storage and CaCl2

43. ANOVA for total soluble solids of apple cultivars as affected by 190 storage and CaCl2

44. ANOVA for total sugar (%) of apple cultivars as affected by 190 storage and CaCl2

45. ANOVA for TSS/Acid ratio of apple cultivars as affected by 190 storage and CaCl2

46. ANOVA for ascorbic acid (mg/100g) of apple cultivars as 191 affected by storage and CaCl2

47. ANOVA for firmness (kg/cm2) of apple cultivars as affected 191 by storage and CaCl2

48. ANOVA for fruit density (g/cm3) of apple cultivars as affected 191 by storage and CaCl2

49. ANOVA for bitter pit (%) of apple cultivars as affected by 192 storage and CaCl2

50. ANOVA for percent soft rot of apple cultivars as affected by 192 storage and CaCl2

CHAPTER 1: GENERAL INTRODUCTION

The apple Apple (Pyrus domestica L) is one of the most important tree fruit of the world. The apple was cultivated in Greece around 600 BC or earlier. It is a highly nutritive fruit which is a rich source of sugars 11%, fat 0.4%, protein 0.3%, carbohydrates 14.9%, vitamins and minerals. A 100 g fresh apple contains, water 84.7%, fibre 0.8 g, carbohydrates 13.9 g, proteins 0.4 g, lipid 0.3 g, ash 0.3 g, vitamin C 8 mg/100gm, sodium 0.3 mg/100g, potassium 145 mg/100g, calcium 7 mg/100g, magnesium 6 mg/100gm, iron 480 μg/100g, Phosphorus 12 mg and Iodine 2 μg (Hussain, 2001). Due to its high nutritional value, it ranks third in consumption after citrus and banana (Bokhari, 2002).

Status of apple in Pakistan In Pakistan its cultivation is limited and restricted to the northern hilly tracts of Punjab and KP, and the Quetta region of Balochistan. Currently, the apple is grown over an area of 11.13 thousand hectares with a total production of 437.39 thousand tons in Pakistan (MINFA, 2008-09). In KP, the apple plantation is distributed in Swat, Dir, Mansehra, Parachinar, Chitral, Hunza, North and South Waziristan Agencies. District Swat, with an area of approximately 4000 square miles with in the Malakand Division, is the most important of all the apple producing districts of Khyber Pakhtunkhwa followed by the districts of Mansehra, Dir, Abbottabad, Chitral and Hunza (Bokhari, 2002; Ali et al., 2004). Increased production of apple by extending its cultivation of apple in low altitude areas of Pakistan is limited by its high chilling requirements (Janick, 1974).

Climatic requirements Due to its chilling requirements, it grows best in relatively cooler climates than other deciduous fruits (Westwood and Chestnut, 1964). can endure quite low temperatures, but temperatures of –30oC and rapid fluctuation in winter from relatively warm to extremely cold temperatures are harmful (Bokhari, 2002). The apple gives better yield in relatively long, cool and slow growing season, the type of climate which usually prevails at altitudes of 1700-2500 m. Soil requirements

Apple trees grow well in a wide range of soil types. They prefer soils with a texture of sandy loam to a sandy clay loam. Good soil drainage is also critical for successful apple production. Ideal soil pH for apple tree is near 6.5 (Gao, 2001). The growth and development of apple tree is adversely affected by water logged soils, rising water tables into the root zone even for a short time, soils having hardpan and shallow soils (Chaudhary, 1994).

Common apple cultivars grown in Pakistan Various cultivars of apples which are being grown in Pakistan include Top Red, Red Spur, Red Delicious, Golden Delicious, Super Gold, Red Chief, Apple Elite, Stark Crimson, Oregon Spur, Red Rom Beauty, Royal Gala, Mondial Gala, And Double Red (Chaudhary, 1994).

The need of apple storage Being in high demand throughout the year, the apple is generally stored in cold storage. In relatively cold climates, simple warehouses may be effective for short term storage (Mitropoulos and Lambrinos, 2000) but cold storage is required for long term storage and quality retention. It is estimated that about 17% of apples produced in Balochistan are lost during postharvest operations (Shah et al., 2002). In Pakistan, apples kept under the conditions of cold storage for 22 weeks, losses were found to be 28 percent (Ilyas et al., 2007).

Postharvest losses Postharvest and storage losses may vary among different cultivars (Golias et al., 2008), due to internal quality characteristics such as titratable acidity, soluble solids, fruit flesh firmness, ethylene production and weight loss in cold storage (Golias et al., 2008), which may in turn influence the texture and storage performance of apple cultivars (Perring, 1989; Hoehn et al., 2003). Cultivars may also differ in fruit physiology and anatomy (Saleh et al., 2009), including ethylene production, texture, fruit flesh firmness (Knee et al., 1983; Larrigaudiere et al., 1997; Stow et al., 2000; Johnston et al., 2001; Nilsson and Gustavsson, 2007) and water loss during storage (Khan and Ahmad, 2005). The physical properties such as fruit flesh firmness (Wilson and Lindsay, 1969; Hudson, 1975; Zaltzman et al., 1987), density and juice (Wolfe et al., 1974; Zaltzman

et al., 1987) of apple fruit is a measure of dry matter (Jordan et al., 2000) and may vary with maturity and during storage (Mitropoulos and Lambrinos, 2000). Significant variations in physical characteristics have been reported for different cultivars and correlated with storage performance of apple (Karathanos et al., 1995), gas exchange (Ho et al., 2010) and subsequent storage life (Meisami et al., 2009). Postharvest losses also depend on production area and market distance and the time of transportation. It is reported that the total losses in the apples transported from Quetta, Swat and Murree to Faisalabad market during the months of August, September and November were found to be 23, 20 and 25 percent respectively (Ilyas et al., 2007). The postharvest losses may also depend on storage conditions. Among the external conditions, temperature and relative humidity during postharvest handling operations are the most important factors influencing the storage performance of apple (LeBlanc et al., 1996), which affect the fruit flesh firmness, juice content, weight loss, pH, soluble solids content (SSC), and other quality (Tu et al., 2000).

Extending the storage life of apple The apple fruit is in high demand throughout the year and hence a considerable quantity is generally stored in cold storages in Pakistan. Apple being a perishable commodity is prone to qualitative and quantitative losses after harvest. The losses may occur during postharvest operations or storage which could be as high as 17% (Shah et al., 2002) or even greater (Ilyas et al., 2007). The postharvest quality and losses during storage may depend on cultivar (Watkins, 2003; Saleh et al., 2009), cultural practices (Tomala, 1999), nutritional status (Hernandez et al., 2005), harvesting stage (Ferguson and Watkins, 1989) and storage condition (LeBlanc et al., 1996). Thus, the selection of best adopted cultivars (Ferguson and Watkins, 1989), optimum pre and postharvest management (Conway et al., 2002) and optimum storage conditions (Lau, 1992) can be used to minimize postharvest losses as well as increased the storage life of apple fruit (Mahmud et al., 2008; Gupta and Jawandha, 2010).

Low temperature storage Apple fruits are kept in cold storage after harvest to preserve their quality. Low temperature plays main role in slowing the degradation of apple fruit quality during storage, depending on the sensitivity of particular cultivars to chilling injury. Apples,

like other climacteric fruits, display abrupt increase in ethylene production during ripening that lead to changes in texture, fruit flesh firmness, color etc. Endogenous ethylene plays key role in apple softening and rapid fruit softening of several cultivars is associated with a rapid rise in ethylene production (Knee et al., 1983; Larrigaudiere et al., 1997; Stow et al., 2000; Johnston et al., 2001; Nilsson and Gustavsson, 2007). The low storage of apples offers the prospect of preventing or delaying softening and improving the texture and quality of the fruit available to consumers (Golias and Letal, 1995).

Harvesting stage and storage life In Pakistan, the apples are harvested at edible maturity for both fresh market and storage. Fruit harvested at this stage have advanced in maturity and are more prone to mechanical injury, have short storage life and greater susceptibility to pathogens and physiological disorders (Juan et al., 1999). In addition, careless harvesting characterized by immature and over mature fruit, is another serious cause of post harvest losses (Ingle et al., 2000). Being a climacteric fruit, the apple can be harvested at physiological maturity (Roth et al., 2005), stored to catch good price in the market (Sayin et al., 2010). In general, apple fruit harvested at immature stage have poor color and flavour and can be more susceptible to physiological disorders such as bitter pit and superficial scald (Kader and Mitchell, 1989; Kvikliene, 2008). By contrast, fruit harvested over-mature tend to be soft and easily damaged during post harvest operations (Ingle et al., 2000). Such fruits are more susceptible to diseases and physiological disorders as well as quality deterioration during or after storage (Ingle et al., 2000; Hribar et al., 1996).

Calcium treatment and storage life Several physiological disorders and diseases of apple fruit during storage are related to the calcium content of fruit (Huder, 1981; Shear, 1975). Calcium deficiency results in economic losses in fruit crops, including apples (Dyson and Digby, 1975; Wilkinson and Fidler, 1973). It helps in regulation of metabolism in apple fruit, and adequate concentration maintain fruit flesh firmness and minimize the incidence of physiological disorders like water core, bitter pit, and internal breakdown (Bangerth et al., 1972; Faust and Shear, 1968). The increase in calcium is generally delayed the ripening and fruit maintain their quality during prolong storage. The application of

calcium also reduced the incidence of storage decay (Conway, 1982; Sharples and Johnson, 1977). Thus it is imperative that compositional changes in apple fruit of different cultivars are evaluated over a range of storage duration to standardize the storage duration, harvesting time and calcium concentration for each cultivar.

General objectives:

1. Evaluate the quality of different apple cultivars 2. Evaluate the storage performance of apple cultivars 3. Examine the changes in internal quality characteristics during storage of apple cultivars 4. Determine the influence of harvesting stages on the quality and storage performance of apple cultivars

5. Evaluate the influence of CaCl2 application on the quality and storage performance of apple cultivar Red Delicious

6. Optimize the CaCl2 concentration and dipping duration to minimize the quality losses during storage in apple cultivar Red Delicious

CHAPTER 2: REVIEW OF LITERATURE

The apple The Apple (Pyrus domestica) is one of principal fruits, grown in temperate region of the world. It has beautiful and colourful appearance, crispy flesh, pleasant flavour and sweet taste that attract the consumers and fetch good price. In Pakistan apples are grown in temperate region of the country such as Murree Hills (Rawalpindi), part of Peshawar region, Northern areas, Kashmir and Quetta (Ali et al., 2004). Kashmiri, Kashmir Amri, Kandhari, Kulu, Kalat Special, Red , Golden Delicious, Banki and Sky Spur varieties are grown in temperate regions whereas, Tropical Beauty, Einsheimer and Enna produce good quality fruit at lower altitudes (Chaudhary, 1994). About 80% of the total apple production of Pakistan is contributed by Kalat, Killa Saifullah, Loralai, Mastung, Pishin, Quetta and Ziarat districts of Balochistan province. The area under this fruit has increased five times during 80 and 90 decades of 20th century (MINFAL, 1999). The apple is a rich source of nutrients and contains, water 84.7%, 0.8 g fibre, 13.9 g carbohydrates, 0.4 g proteins, 0.3 g lipid, 0.3 g ash, vitamin C 8 mg/100gm, sodium 0.3 mg/100g, potassium 145 mg/100g, calcium 7 mg/100g, magnesium 6 mg/100gm, iron 480 μg/100g, Phosphorus 12 mg and Iodine 2 μg (Hussain, 2001). Besides fresh consumption of apple fruit, it is used in many products like, jams, jellies, marmalades, muraba, salads, sandwich, filling, snacks, in many dishes, puddings, sweet meats, pickles and other preserves include pie filling, slices and sauces. In foreign countries fermented is used for alcoholic purposes. Sour varieties of apple are used for the preparation of fermented apple juice as (Hulme, 1970). Perez et al. (2001) reported that the US per capita consumption of apples has risen over the past three decades, with consumption of processed apple products exceeding consumption of fresh apples in the last 20 years. While fresh apple consumption remained fairly stable, the largest increases in processed per capita use during the 1990s were for juice, frozen, and dried products. Physical characteristics of agricultural products are the most important parameters to determine the proper standards of design of grading, conveying, processing and packaging systems (Tabatabaeefar and Rajabipour, 2005). Among these physical characteristics, mass, volume and projected area are the most important ones in determining sizing systems (Khodabandehloo, 1999). Quality differences in fruits can

often be detected by differences in density. When fruits are transported hydraulically, the design fluid velocities are related to both density and shape. Postharvest evaluation gives possibilities for delivering a high quality product and a basic understanding of apple texture is necessary for the development of technology for postharvest evaluations (Ioannides et al., 2007). Mechanical properties of the tissue determine the susceptibility to mechanical damage that can occur during harvest, transport and storage and that eventually leads to a profound reduction in commercial value (Oey et al., 2007). Mechanical properties such as failure stress and strain as well as modulus of elasticity can also be used to evaluate the behavior of the fruits mechanically under the static loading. Firmness or hardness is another important attribute of fruits and it is often used for fruit quality assessment (Vursavus et al., 2006).

Chemical properties Information regarding chemical properties of fruit is crucial in processing it into different foods (Vursavus et al., 2006). The sugars content, sucrose, glucose, fructose, and sorbitol, in fruit flesh contribute to the fruit sweetness, and are one of the characteristics of fruit quality and market value. The apple fruit accumulate starch at the early stages of maturation that is later on hydrolyzed to sugars (Magein and Leurquin, 2000). Golias et al. (2008) stored the apple five cultivars in cold storage and studied the chemical attributes for titratable acidity, soluble solids, firmness, ethylene production and weight loss for 100 days. The changes in titratable acids, ethylene production and loss of firmness significantly differentiated the cultivars, although Golden Delicious, Reinders and Resista still could not be completely separated. Total soluble solids and loss in weight did not contribute to the discriminant resolution. Khorshidi et al. (2010) studied the postharvest quality of Red Delicious apple under different temperatures (0, 5 and 12°C) for one month. They found that the fruit diameter, fruit weight, volume, firmness, total titratable acids (TTA), total soluble solids (TSS), elements of sodium and potassium, marketable quality and color surface were significantly affected by different storage temperatures. However, the Red Delicious apple fruit stored at temperature 0oC maintained the better quality attributes. Rutkowski et al. (2008) used optical reflectance spectrometry method for the measurement of chlorophyll and evaluate other quality parameters in „Golden Delicious‟ apples. They reported that fruit firmness, chlorophyll content and

acidity were decreased during vegetative and postharvest period. Significant interaction was observed in chlorophyll content, titratable acidity and fruit firmness. Thammawong and Arakawa (2010) harvested mature and immature apple fruits and treated them with 1-MCP and ethylene for evaluating their response on sugar accumulation. Immature fruits treated with ethylene showed decreased amount of starch while total sugar content was not significantly affected. Ethylene and 1-MCP did not significantly affect the ripening aspects of immature fruit. They reported inverse correlation of sugars accumulation with ripening properties and starch hydrolysis in „Tsugaru‟ fruit during storage.

Physical variations The physical characteristics of apple fruits are important for their storage as well as processing properties. Significant variations have been reported in physical characteristics among apple cultivars (Weibel et al., 2004). Appearance, tastiness and texture are the main determinants of product‟s quality accepted by consumers. According to Surmacka-Szczesniak (2002) texture is an indicator of structural and mechanical properties of food products and determines consumer‟s acceptability. Fruit firmness is one of the basic criteria of fruit texture estimation and in some countries a specific degree of firmness is included in primary parameters for marketing (Hoehn et al., 2003). The fruit firmness depends on fruit density related with the quality and storage performance of apple fruit (Amarante et al., 2000). However, the softening rate has also been reported to vary from cultivar to cultivar, depending on the presence and expression of genes which regulate the activity of hydrolytic enzymes (Ingle et al., 2000; Konopacka and Plocharski, 2002; Johnston et al., 2001). Chang-Hai et al. (2006) evaluate the response of peach fuirt firmness to different levels of temperature during storage. They recorded the delayed softening of peach fruit and inhibition of changes in cell wall and materials at low temperature of 5°C. They reported that softening of fruit cell wall was due to increased activities of cell wall polysaccharide-related enzymes at higher temperature. Sakiyama and Nakamura (1980) subjected cucumber fruits to different levels of temperature under dry and wet conditions with maintaining their specific gravity at constant while volume was at high. The specific gravity and volume of cucumber fruits which were in wet conditions was changed at 20°C and became similar to fruits stored in dry

conditions. A non-significant change was observed in volume and specific gravity in fruits stored at 10°C in humid conditions, with a little change in fresh weight. By removing wet conditions their specific gravity and volume was changed at higher rate. Meisami et al. (2009) observed the physical characteristics of apples having different diameters. Average mass and volume were 74.87 g and 104.5 cm3 respectively while apparent density and density were 0.2401 g/cm3 and 0.7427 respectively. Porosity of apples having different diameters was 57.24, 54.13 and 50.17 percent and their Average packaging was 0.45. Kheiralipour et al. (2008) evaluated different physical and chemical attributes of two apple cultivars. They reported that two apple cultivars were significantly different in different physical characteristics at the one percent probability level. Nislihan and Celik (2006) stored the apple cultivars in controlled environment with 0oC temperature and 85-90% relative humidity. Then heat treatments were given for 4 days at 38oC to these cultivars. High weight loss was observed in cultivars with heat treatment which was found effective for firmness during storage. The heat treatment increased the ripening by accelerating the process of respiration and starch degradation. Crouch (2003) treated apple cultivars with Smart Fresh and reported significant changes in fruit firmness and incidence of bitter pit. Apple cultivar Royal Gala showed highest firmness (5.7 kg/cm2) which was followed by cultivar Red Delicious (5.6 kg/cm2) while lowest firmness (4.9 kg/cm2) was recorded in Golden Delicious. Minimum bitter pit incidence was reported in cultivar Red Delicious. The physical properties of the fruit determine the diffusivity of water gases through the fruits. Thus, influences the availability of oxygen for respiration and water loss. Ho et al. (2010) used permeation diffusion reaction model to study the gas exchange of apple fruits. They measured the gas respiration parameters and exchange properties of the fruit organ tissues. The measurements revealed the existence of metabolic gases in apple fruit. Large potential for controlled atmosphere (CA) storage was recorded in

Jonagold while showed low diffusion properties. Kanzi had less O2 anoxia at CA storage compared with Braeburn. Karathanos (1995) used different concentrations of sucrose for determining the air drying kinetics of fresh and dehydrated apples fruits. The samples that were pre-treated with concentrated solutions of sugar of 45% significantly decreased the diffusivity. The low diffusivity helps in storage stability and for better utilization of fruits.

Postharvest problems

The apple fruits are characterized by relatively low rates of respiration 5-10 mg CO2 kg-1h-1) and high rates of ethylene production (10-100 µl kg-1h-1) as well as sensitivity to ethylene (Kader, 2002). Therefore, the apple fruits are prone to significant postharvest losses during postharvest handling and storage. According to (LeBlanc et al., 1996), the fruit production chain, including harvest, storage and distribution are generally not perfect with 90% of fresh apples stored under improper conditions, especially during the summer period. It is found that some cultivars are more susceptible to decay than the others. Thus the incidence of different pathogens on apple fruit such as blue mold, gray mold, bull‟s-eye rot, and mucor rot is dependent on cultivar (Spotts et al., 1999). Hence apple cultivars are generally selected for resistance to certain postharvest diseases. For example, „Royal Gala‟ is extremely resistant to wound pathogens, „‟ to skin punctures, and „Braeburn‟ to infiltration of fungal spores into the core (Spotts et al., 1999). The onset of ripening and senescence in various fruit and vegetables renders them more susceptible to infection by pathogens (Kader, 1985). Extended storage of apple fruit may cause enzymatic browning accompanied by unpleasant colors and flavors and a loss of nutrients. The browning of apple fruit during storage depends on polyphenol content and polyphenoloxidase (PPO) activity (Goupy et al., 1995), an enzyme that catalyses the oxidation of polyphenols to their corresponding quinones (Rocha and Morais, 2001). Shah et al. (2002) analyzed quality and marketing of apple produced in Balochistan. There are two cold stores in Balochistan which have the capacity of 700 tones. Because of tight packaging and wooden crates 17 percent of apples were found damaged in cold storage, while 12 percent were unconsumed. in Quetta market during the month March 70% of apples were shin kulu while the rest were Gala, Tore kulu,, Mashadi and Kashmiri. The apple variety Shin kulu resulted longer shelf life as compared to other varieties because of firmness in texture. Ilyas et al. (2007) estimated the losses of apples during transportation in different months. When apples were kept in cold storages then losses decreased to 28 percent because certain types of fungus related infections were controlled. The pathogenic fungi in both inoculated and non inoculated apple fruits were Rhizopus nigricans, Aspergillus niger and Alternaria tenuis, while the rottening of banana fruits were due to a number of fungi and fumigates was also pathogenic to both injured and non injured banana fruits. Tu et al.

(2000) examined apple cultivars at different RH conditions keeping in view the quality of apples. Depending on the cultivar some apples at RH 95% and 20°C develop mealy texture. The 65% RH is typical and 30% is a low RH for increasing shelf life. The firmness of apples for both apple cultivars became decreased with a slower rate at higher RH, while weight loss became higher at low RH. Higher levels of RH was helpful in maintaining firmness of apples and in decreasing weight losses but it promote mealy texture at 20°C. Scheper et al. (2007) evaluated the effect of washing and unwashing treatments on post harvest apples in controlling storage rots in injured and uninjured fruits. Washing of apples in high fungal populations significantly affected the punctured fruits as compared the uninjured fruits while no significant difference was observed in punctured and uninjured fruits when washed with water containing less population of fungi. Penicillium spp. was the main causal agent of rots while non significant effect was observed in yeast rot incidence. Pesis et al. (2009) treated apple fruits with ethylene and then stored at cold storages to observe the ripening process and superficial scald development of climacteric fruits. The bitter pit and development of superficial scald was greatly suppressed by cold storage at 0oC. the transformed lines of 103Y, 130Y and 68G a little amount of ethylene in cold storage at 0oC during the first 3 months and then little increase at 20oC, while the untransformed fruits significantly increased the ethylene production at cold storage which was dramatically increased at 20oC and respiration was not significantly affected. The line 68G produced greater amount of both (E, E) alpha-farnesene and (Z, E) alpha-farnesene as compared to yellow and green fruits in all lines after 2 months at 0oC. (E, E) alpha-farnesene produced 100 times more than (Z, E) alpha- farnesene in all lines. The severity of superficial scald were more in untransformed GS apples whereas lines 103Y and 68G show less severity while line 103Y was free from any scald. Green fruits were severely affected from superficial scald as compared to yellow fruits. The production of 6-methyl-5-hepten-2-one (MHO) was also higher in these lines which is a major oxidation product of (E, E) alpha- farnesene. Superficial scald and MHO were absent in line 130Y. they reported that transgenic apples produce alpha-farnesene which then oxidizes to MHO and free radicals and develop superficial scald.

Apple cultivars and storage performance A large number of apple cultivars have been developed and considerable differences have been reported in their storage performance. Saleh et al., (2009) stored four apple cultivars in two seasons at 0ºC temperature and 85-90% relative humidity in different durations. Highest weight loss was recorded in Gala while least weight loss was observed in Star Cremson. Golden delicious showed highest fruit firmness while lowest firmness was observed in Star Cremson and Starking Delicious. All apple cultivars were significantly affected in all parameters during cold storage. Apple cultivar Star Cremson showed highest fruit storability. Ali et al. (2011) examined chemical changes in apple pulp during storage. They reported that ascorbic acid content, pH and sugar acid ratio was decreased while TSS and titratable acidity was increased in 90 days of storage. Treatments and storage intervals significantly affected the physico-chemical properties of apples. Omaima et al. (2007) treated pre-harvest apple cultivar with boric acid and calcium chloride as a foliar spray to decrease the incidence of Bortytis cinerea, a main causal agent of fruit rot. The combined treatments of foliar spray significantly increased total soluble solids, fruit firmness, total anthocyanine and total sugar, while decreased fruit rot decay percentage, weight losses percentages and total acidity at 5°C and cold storage for 60 days. Kvikliene et al. (2006) evaluated the pre and post harvest chemical changes in apple cultivar „Auskis‟. Least weight loss was observed in apple cultivar harvested at optimum maturity. Fruit firmness was decreased with late harvesting. Positive correlation was observed between firmness at harvest and post-storage acidity and negative correlation was observed in firmness at harvest and post-storage sugar/acid ratio. Post-storage sugar/acid ratio and post-storage soluble solid content were correlated to soluble solids content at harvest. They reported that optimal harvest time is in between of 114 and 121 days for apple cultivar „Auskis‟ after full bloom. Markuszewski and Kopytowski (2008) grafted different apple cultivars on each other and applied soil cultivation in six manners in rows. After the harvest and storage period, fruits were contained less ascorbic acid, dry matter and organic acids and more simple sugars and total sugars. The cultivar „Szampion‟ grafted on MM.106 showed best results for all parameters. The manner of soil cultivation significantly affected the apple cultivars for most of the parameters while the best results were obtain with manure and polypropylene fabric. Eugenia et al. (2006) compared the four apple cultivars in both traditional and refrigerating storages. Refrigerating storage

showed best results as compared to traditional storage. Refrigerated storage resulted lowest dehydration in Wagener Premiat varieties while highest water loss was recorded in . Wagener Premiat varieties also resulted better qualities regarding ascorbic and acid total sugar as compared to other varieties. They reported that apples should be stored in controlled atmosphere (CA) storage to keep the apples in optimum conditions. Ali et al. (2004) stored five apple varieties in ordinary storage at room temperature of 25oC. An increase was observed in reducing sugar while a decrease was observed in non-reducing sugar and total sugars were increased with the prolonged storage condition. Acidity was non significantly affected while total soluble solids significantly increased during storage at room temperature. A decrease was observed in Vitamin C during storage. They recommended Golden Delicious and Amri cultivars of apple for storage to fetch good market price. Khan and Ahmad (2005) stored five apple cultivars under ordinary storage conditions at room temperature in September. The first two weeks of storage did not significantly affected the apple cultivars while gradual change in weight loss and fruit frimness was observed with four weeks of storage. Apple cultivar Amri showed highest weight loss during six weeks of storage while the cultivar Kalakulu resulted least weight loss. Dobrzanski et al. (2001) established sorting line for sorting and sizing of apples. From outlets of sorter apples of different sizes were analyzed for their weight, size and colour for assigning a fruit quality index. For achieving final fruit quality index some nutritional values of apple like L-ascorbic acid and reducing sugar were also determined. Eisele and Drake (2005) compared 175 apple varieties collected from several geographical areas of USA in terms of their pH, Brix, glucose, fructose, sorbitol, citric, calcium and sodium levels with existing compositional database values. The juices obtained from apple varieties were highly variable in terms of their phenolic compounds. Some of the characteristics were highly matched with one another such as the phloridzin and chlorogenic acid were in same levels in all varieties of apples while arbutin was in not measurable levels. They reported that the data developed from different apple cultivars is useful with other databases for the development of apple commercial varieties in future to meet the consumer requirements.

Harvesting time To ensure maximum storability, apples should be picked when mature, but not fully ripe. If apples are picked when they are too ripe, physiological processes are underway which complicate storage, even under optimal conditions (Ingle et al., 2000). Apples picked at right stage have the organoleptic qualities which enable them to survive more than six months of storage. Apples which were harvested the earliest were firmest both before and after storage, but lost a greater percentage of their firmness during storage. Apples harvested 100 days after full bloom (DAFB) had a firmness of 10.2 kg at harvest and 5.0 kg after storage, and lost 51% of their initial firmness. Apples harvested 128 DAFB had a firmness of 8.2 kg at harvest and 4.7 kg after storage, and lost 43% of their initial firmness. Apples harvested 114 and 21 DAFB lost only 41% of their initial firmness. This agrees well with an earlier study on fruit softening in other cultivars (Meresz et al., 1993). Fruits that are picked before physiological maturity will not ripen satisfactorily (Robertson et al., 1990), while those harvested at more mature stage have shorter shelf life (Meredith et al., 1989) and did not ship well because of reduced shelf life (Murray et al., 1998). Peaches, if harvested too early are small, very firm in texture, with low sugars, reduced flavour and colour while the later picked fruits are very soft, high in sugar and water content and all the physiological processes which complicate storage are underway. Once the fruit ripens, senescence begins; physical and chemical changes continue after optimum ripeness is reached including further softening, loss of desirable flavor and complete breakdown (Kader and Mitchell, 1989). Gupta and Jawandha (2010) studied the response of peach cultivar „Earli Grande‟ to three stages of fruit harvest and evaluated their physical and chemical characteristics to cold storage of 0-20oC temperature 85-90% relative humidity for 21 days. The fruit quality parameters were significantly affected by different stages of fruit harvest. An increase in physiological loss in weight, acid ratio, spoilage, TSS and anthocyanins was observed with the delay in harvesting stage and increase in storage time. With the advancement in maturity and storage duration a linear decline in Vitamin A content was observed. A gradual decrease of reducing sugars was observed in fruits with the increase in storage period picked after optimum maturity. They reported that peach fruits harvested at optimum maturity retained maximum TSS acid ratio and could be stored for three weeks in cold storage.

Lafer (2006) investigated the response of apples to different harvesting dates while applying treatments of AVG before harvest and 1-MCP after harvest. Storage in controlled atmosphere treated with 1-MCP and AVG showed good titratable acidity and firmness while delayed the fruit softening. Over matured fruits lost more acidity and firmness as compared to those fruits which were harvested at optimal maturity stage. AVG and 1-MCP treatments also affected the amount of Total soluble solids. Storage for more time showed CO2 damages, senescent scald and fungal decay. Maximum incidence of internal browning was observed in over ripeed apples treated with 1- MCP. McLellan et al. (1990) harvested apple fruits at three different stages. The harvested fruits were at cold storages with a 95% RH. The slices of apples were taken from different treatments for analyzing their Brix/acid ratio. Raw slices of apples were analyzed though Sensory analysis. Slice firmness was due to CA delay and harvesting date. With the delay of storing at CA storage the un-blanched raw slices, showed softening, while late harvest also resulted higher softening. Blanching of slices greatly increased the softening. A significant increase in apple flesh browning was recorded at delay in storage at CA and due to later harvest. There was no significant difference in acceptability rating of raw slices of apples before freezing. Echeverria et al. (2002) evaluated the response of fruit quality and production of aroma of apples to influence of storage conditions and harvest date. Fruit firmness, soluble solids, titratable acidity, physiological disorders, skin color and aroma production was measured after 3, 5 and 7 months of storage. Controlled atmosphere maintained good quality of fruits as compared AIR atmospheres. Apples treated with ULO1 maintained good firmness in storage conditions as compared to other treatments. AIR atmosphere maintained the more aroma of apple. By passing of more time, decrease in aroma was recorded. Storage of apples in ultra-less O2 atmospheres showed less aroma production treated with ULO2. Erkan and Pekmezcu (2004) studied the effect of harvest dates (15 days interval) on superficial scald development and postharvest quality in „Granny Smith‟ apples ( domestica) stored at 0oC with 90% relative humidity for 8 months. A significant variation was observed for weight loss, soluble solids, titratable acidity and flesh firmness among the different harvest dates. Increased soluble solids were achieved with Delay harvest. Flesh firmness, titratable acidity and soluble solids remained at acceptable levels regardless of harvest dates and storage durations. Early harvested fruits were decayed at a lower rate. The „Granny Smith‟ apples could be stored for 8 months with minimal scald

incidences (0% to 14.2% depending on storage length). Hernandez et al. (2005) harvested apple (Malus domestica) of the fruit at two or three different maturity stages stored at 33 ºF in air or in controlled atmosphere (CA). Fruit stored in CA conserved higher firmness and produced less CO2. Internal browning was not seen in fruit stored in air, but appeared in fruit after two months storage in CA. The incidence did not increase after longer storage times. It did not affect the incidence of internal browning, but DPA inhibited internal browning completely. A mineral analysis of the apple flesh showed differences among the seasons. Concentrations of calcium (Ca), boron (B) and magnesium (Mg) were significantly higher, corresponding with a lower incidence of internal browning. Tomala (1999) investigated the effect of several renowned factors on the quality of pome fruits at harvest and following storage. Sensitivity to low calcium and susceptibility to physiological disorders during storage was observed by the apple fruits. Calcium content of apple at maturity was influenced by environmental and cultural factors. Important factors (fertilization, pollination, seed number and fruit set) played a crucial role in fruit quality after breakage of dormancy and appearance of bloom. Fruits, which develop from terminal flowers, are richer in calcium than those developing from lateral ones. The location of fruits in the tree crown is also influencing calcium concentration, and incidence of physiological disorders; more fruits are affected in the upper parts of canopy. Calcium treatments reduced the occurrence of physiological disorders. Apples low in calcium showed an earlier onset of the endogenous ethylene climacteric as fruits mature on the tree as compared to early blooming ones. Fruits from late blooming flowers produced less ethylene, exhibited a lower starch index and developed superficial scald during storage. Steenkamp et al. (1983) recorded that bitter pit tissue had a higher concentration of calcium, potassium and magnesium than sound tissue as well as higher concentrations of oxalic and citric acid but a lower concentration of malic and succinic acid. It appeared that localized excessive concentrations of oxalic and citric acid could induce bitter pit. Cocucci (1983) reported the mechanism energy dependent able to secrete proton, linked to cation influx in plasmalemma of apple fruit (cv. Granny Smith). This helps in uptake of organic and inorganic compounds by apple cells during ripening. The pitted fruits had 60% lowered proton secretion as compared to fine ones but similar transmembrane electric potential at low k+ level. The pitted fruits had lower Ca2+ content as compared to the fine ones. Calmodulin level was 40% higher in the

pitted apple fruits than in the sound ones. This result indicated an involvement of the calmodulin- Ca2+ system in the bitter pit. Juan et al. (1999) investigated the effect of harvest date on storage ability of 'Golden Delicious' apple and cold stored for seven months. Quality indices (soluble solids content, flesh firmness, acidity and starch index) were determined weekly for one month before the first harvest date and upon removal from storage. Fruit susceptibility with increasing maturity was inoculated with Penecillium expansum, keeping in cold storages for five weeks. After removal from cold storage, superficial scald and moisture loss incidence were higher on fruit picked earlier. Bitter pit occurrence was also observed. The percentage of marketable size fruit and disease severity increased with harvest date. Starch index was significantly correlated to acidity, soluble solids and firmness, suggesting that it could be used to predict fruit quality after cold storage. Kvikliene et al. (2008) studied the effect of fruit maturity on apple fruit cv. „Ligol‟ storage ability and rot development. Fruits for storage were harvested 5 times at weekly intervals before, during and after predictable optimum harvest date. The fruits quality parameters changed according to harvest date. Later harvested fruits were softer. Content of soluble solids did not depend on harvest time. Fruit storage ability was closely connected to fruit maturity. The best quality apple fruits one week before climacteric peak were picked after 180 days of storage.

Calcium and postharvest performance According to Conwey et al. (1982) fruits and vegetables require additional cost in value addition, when they are moved from the field to the consumer. Thus, it is an economic necessity to decrease by extending the storage life. While several treatments e.g. heat, chemicals, and irradiation can be used to reduce incidence of microorganisms, but injury to fruit and consumer‟s concerns about chemical residues or ionizing radiation requires the development of alternative methods of protection. Calcium is an important second category macro-nutrient which is involved in variety of different function. Calcium helps to regulate the metabolism in apple fruit, and adequate concentration maintain fruit firmness, delays fruit ripening, lower the incidence of physiological disorders such as water core, bitter pit, and internal breakdown (Bangerth et al., 1972; Faust and Shear., 1968; Mason, et al., 1975; Reid and Padfield., 1975) and suppress Erwinia carotovora (Jones) incidence on apple fruits (Conway, 1982; Sharples and Johnson., 1977).

Mahmud et al. (2008) treated Papaya (Carica Papaya L.) fruits with 1.5, 2.5 and 3.5% solutions of calcium chloride by dipping and vacuum infiltration (33 Kpa) or untreated (0%) as control to study the storage life and postharvest quality characteristics. The postharvest infiltration of calcium at 2.5% has the potential to control disease incidence, prolong the storage life and preserve valuable attributes of postharvest papaya, presumably because of its effects on inhibition of ripening and senescence process and loss of the fruit firmness of papaya. Kadir (2005) sprayed

„Jonathan' apple trees at three commercial orchards with calcium chloride (CaCl2) solution containing 3.2 g/L, starting when apple sizes were between 0.9 and 1.6 cm average diameters. Apples were stored for two and four months in regular atmosphere storage at 2°C (36°F). Apples stored for two months had better quality than those stored for four months. Depending on the location, five to eight CaCl2 applications and two to seven applications were necessary to retain an average of 26% of fruit firmness and an average of 35% of the SSC/TA, respectively, in the two-month storage. At least seven applications were required to retain an average of 29% of fruit firmness of apples stored for four months. Six to seven applications of CaCl2 retained fruit weight by 22 to 33% more than the non-treated control apple. In general, CaCl2 was beneficial for storage quality of 'Jonathan' apples in Kansas. Trentham (2008) stored the apple fruits at 0oC after dipping for 2 minutes in 0, 2%, 4%, or 6% solution of CaCl2 at 0 or 68.95 kPa. He recorded the data for different parameters with the interval of four months. Paraffin sections were stained with an aqueous mixture of alcian blue 8GX, Safaranin 0 and Bismark brown Y, or with theperiodic acid-Schiff (PAS) reaction. No histological difference was observed in fruit treated with 2%

CaCl2 compared with those pressure-infiltrated with greater amounts of Ca. Fruits pressure-infiltrated with 6% CaCl2 exhibited the greatest amount of flattened epidermal cells and hypodermal cavities. Cuticles were also affected at the higher

CaCl2 treatment levels (with regard to staining with Bismark brown), becoming more condensed and uniform. Cuticle and hypodermis were stained differentially with PAS in the 6% CaCl2 treatment. All tissues, including the cuticle, were stained magenta red, indicating a possible chemical alteration of the cuticle and the underlying tissue by calcium. Petersen (1980) reviewed the available information regarding calcium (Ca) nutrition of apple trees. In spite of a high Ca content in most orchard soils and a high potential of Ca uptake in apple trees, there is no doubt about Ca deficiency being a causal factor for many disorders in apple fruits. Translocation acts by ion exchange,

mainly in the xylem, and is very slow. Apical meristems and young growing fruits with rapid cell division have a high demand for Ca and depend on a continuous supply. Concurrent with a change in fruit growth from cell division to cell expansion, the Ca intake in the fruits may cease and fruit Ca content may even decrease, as the fruits may then serve to some extent as a source of Ca for growing leaves and shoot tips. Fruits containing Ca at less than 50 mg kg−1 of fresh weight are sensitive to bitter pit and internal breakdown. As dipping the ripe fruit in a Ca solution after harvest often gives the same protection as a Ca spray on the trees, permanent damage in the developing or ripening fruits can be avoided. That means that some of the disorders of the fruits are caused by lack of Ca in the respiratorial and senescence processes rather than in the developmental stage. Conway et al. (2002) suggested that calcium, the most important mineral element determining fruit quality. It seemed to be especially important in apples where it reduced metabolic disorders. Calcium in adequate amounts helped to maintain apple fruit firmness and decreases the incidence of physiological disorders (water core, bitter pit and internal breakdown). Postharvest decay may also be reduced by increasing the calcium content of apple fruit. Directly applied calcium increased fruit calcium content. Both pre- and postharvest calcium treatment methods had inherent problems. Developing a commercially acceptable method of successfully increasing calcium concentration in fruit is a continuing challenge. Freitas et al. (2010) examined that bitter pit, a Ca2+ deficiency disorder of apple fruit (Malus domestica), is a complex process that involves not only the total input of Ca2+ into the fruit, but also a proper Ca2+ homeostasis at the cellular level. The objective of this study was to test the hypothesis that Ca2+ accumulation into storage organelles and binding to the cell wall is associated with BP development in apple fruit. The experiment was carried out on „Granny Smith‟ apples stored at 0oC for 60 days. After storage, fruit were segregated into two lots for analysis, apples with the water-soaked initial visual symptoms of BP and those not showing this symptom. Cytochemical and ultra structural observations showed an accumulation of Ca2+ in the vacuole of individual outer cortical cells of pitted fruit. We also observed an increase in the expression of genes encoding four pectin methyle-sterases, a greater degree of pectin de-esterification and therefore more Ca2+ binding sites in the cell wall, and a higher fraction of the total cortical tissue Ca2+ content that was bound to the cell wall in pitted fruit compared with non-pitted fruit. Cells of the outer cortical tissue of pitted

fruit consistently had higher membrane permeability than outer cortical cells of non- pitted fruit. The results provide evidence that Ca2+ accumulation into storage organelles and Ca2+ binding to the cell wall represent important contributors to BP development in apple fruit. Hayat et al. (2003) investigated the effect of different concentrations of calcium chloride (1%, 1.5%, 2%), paraffin wax coating and different wrapping materials (polyethylene, carton paper) to increase the shelf life and to avoid the postharvest losses of Banky cultivars of apple. All the treatments had a significant effect on the shelf life of fruits. Calcium chloride (2%) proved very useful for reducing weight loss and shrivelling and retained consumer acceptability even after 60 days of storage. Polyethylene packaging stood second position after 2% calcium chloride treatment. Swiątkiewicz and Błaszczyk (2007) studied the effect of late spraying with 0.8% Ca(NO3)2 on calcium content as well as nutrient mutual relations between mineral constituents in „Elise‟ fruit of 5 year old apple tree. In general, foliar spray with calcium nitrate increased Ca concentration in fruits determined both after treatments and after harvest however this effect was modified by weather conditions in particular experimental years. Sprays with calcium nitrate significantly decreased N/Ca and K/Ca ratio in fruits analyzed after treatments as well as freshly harvested fruits, however only in 2005. Castro et al. (2008) studied the biochemical factors associated with a CO2 induced internal flesh browning (FB) disorder of Pink Lady apples (Malus domestica‟). Pink Lady apples were stored in air o or controlled atmosphere (CA) with 1.5 kPa O2 and 5 kPa CO2 at 0.5 C for 2 and 4 months. Both brown and surrounding healthy tissues in apples with FB showed a decrease in ascorbic acid and an increase in dehydroascorbic acid during the first 2 months of storage in CA, the time period when FB developed. Undamaged, CA- stored apples retained a higher concentration of ascorbic acid after 2 months in storage. The level of hydrogen peroxide (H2O2) increased more in the flesh of CA stored apples than in air stored apples, an indication of tissue stress. In addition, concentrations of H2O2 were significantly lower in diphenylamine (DPA) treated apples. Treatment with DPA also inhibited FB completely compared to untreated apples. Poly phenol oxidase (PPO) activity was similar for apples kept in air or CA storage and between undamaged and damaged fruit. The results showed a closer association between FB and the oxidant antioxidant mechanisms such as ascorbic acid, H2O2 and DPA, than to the activity of specific browning enzymes like PPO. Further investigation of the protective effect of ascorbic acid is warranted as is further

research on the underlying causes of apple fruit susceptibility to FB. Biggs et al., (1993) treated apple cultivar Nittany by Alternaria spp with the calcium chloride

(CaCl2) for its efficacy in reducing the incidence and severity of infection. CaCl2 reduced the incidence of rot from 61% in the controls to 27 and 33%, respectively.

Dip treatments alone reduced rot incidence to 17 and 12% for the CaCl2 and liquid

CaCl2 treatments, and seasonal sprays followed by dip treatment reduced incidence to

5%. In postharvest tests, fruit treated with CaCl2 alone and in combination with iprodione exhibited the lowest incidence and severity of Alternaria rot. At harvest, isolation frequency from surface-disinfested fruit averaged 34%. Martin et al. (1960) sprayed different treatments to half-trees of Cleopatra apples, it was shown that magnesium nitrate increased the incidence of pit and calcium nitrate decreased it but increased the calcium content, borax decreased the effectiveness of the calcium nitrate treatment. Magnesium or calcium nitrate, with or without borax, did not affect the potassium, magnesium, phosphorus, or nitrogen content of the fruit cortex. Petersen (1980) conducted an experiment with apple trees, cultivar „Cox's Orange‟, Ca was omitted in the nutrient solution for periods of different length. In the bulked sample, average size of fruits was not considerably affected, but the distribution of small and large fruits changed toward smaller fruits. Concurrently, bitter pit was reduced compared to the control. Average fruit size was not significantly changed, but the distribution was towards larger fruits. Under these circumstances bitter pit was increased. Ca deficiency, caused either by omission of Ca or competition between K and Ca, increased fruit rot, russeting and cracks on the fruits. Short term variations in Ca levels were not seen to produce any measurable response. Perring (1989) examined the development of physiological disorders in particular zones of apples that might be allied to changes in the chemical composition in these and other zones. The flavour and texture of the various zones of the fruit may alter differently during storage because of changes in the distribution of dry matter, water and organic acids.

Bitter pit and calcium Bitter pit is a physiological disorder that appears as depressed brown lesions in the skin of the fruit, located mainly on the calyx end (Ferguson and Watkins, 1989). The disorder is inversely related to the Ca concentration of the fruit and, in general, is directly related to Mg, potassium (K), phosphorous (P), and nitrogen (N) levels in fruit tissues (Fallahi et al., 1997). Summer and root pruning, application of growth

regulators, fruit thinning, Ca fertilization, effect of localized Ca application have shown that the incidence of bitter pit relates to the Ca distribution within the plant than to the total Ca supply from the soil. Because Ca moves mainly through the transpiration stream as the vegetative tissues have less resistance to transpiration, Ca absorbed from the soil will tend to move toward vegetative tissues and away from the fruit (Jones and Higgs, 1982). Reid and Padfield (1975) evaluated the incidence of bitter of apple cultivar „Cox‟s

Orange Pippin‟ after dipping in solution containing 2.5% CaCl2 or Ca(NO3)2. The incidence of bitter pit was reduced considerably when fruit was dipped in solution containing lecithin of egg. Lecithin addition also alleviated the damage often associated with calcium dips and improved control of two other physiological disorders, breakdown and water core. A dip containing lecithin alone was relatively effective. It was suggested that lecithin might assist the movement of applied calcium into apple fruits. Dris and Niskanen (1999) treated five commercial apple (,

Raike, Red Atlas, Akero, Aroma, and Lobo) with preharvest calcium chloride (CaCl2) o sprayed at Ca 2.0 g/l, stored at 2-6 months at 2-4 C and 85-95% RH. Preharvest CaCl2 sprays increased fruit firmness and the titratable acidity but decreased soluble solids, soluble solids/titratable acidity ratio, and the incidence of physiological storage disorders of some cultivars.

Physical characters and apple Jordan et al. (2000) determined the density of unripe kiwifruit (Actinidia deliciosa cv. Hayward) early in storage as a means to find out the current fruit dry matter (DM) and total sugar-plus-starch concentrations, and of predicting DM and soluble solid concentrations later when the fruit fully matured. As fruit taste is related to sugar concentration, and sugars make up the bulk of the soluble solids in fruit. Density to both DM and ripe fruit soluble solids in the composition trial had similar parameter values to those of the survey trial and gave S.E. of prediction of about 0.3% FW. DM levels were about 3.2% FW above the sum of soluble solids and starch concentrations in both ripe and unripe fruit, a difference largely independent of DM concentration. Starch lost during ripening was accounted for by the increase in the glucose and fructose sugar pools, and these two sugars had near equal concentrations at each DM level. Sucrose and minor sugar levels were independent of DM and ripeness. McGlone et al. (2007) measured non-destructive density and Visible-Near Infrared

(VNIR) on yellow-fleshed kiwifruit (Actinidia chinensis) harvested on four occasions across a commercial harvest period. Density measurements were made by flotation and the VNIR measurements using a polychromatic spectrometer system operating over the range 300–1140 nm, although much smaller spectral regions were better for predicting DM and SSC (both 800–1000 nm), or Hue (500–750 nm). Harvest-time and post-storage data sets were formed and used to develop models for predicting harvest-time and/or post-storage quality parameters. The VNIR method proved superior to the density method in every case, especially for DM and SSC predictions where the VNIR method was close to twice as accurate. The VNIR method yielded accuracies (standard errors in prediction) of ± 0.40%, ± 0.71% and ± 1.05° for predictions of harvest DM, SSC and Hue, respectively. Predictions of post-storage DM, SSC and Hue, from post-storage spectra, had improved accuracies of ± 0.24%, ± 0.31% and ± 0.98% respectively. The increased accuracy for SSC prediction, from ± 0.71 to ± 0.31%, is theorized to be a consequence of the VNIR method being better at predicting the total carbohydrate concentration, which comprises starch and soluble sugars in about equal amounts at harvest but is mainly soluble sugar after the fruit ripens during cold storage. That theory was supported by the observation that post- storage SSC predictions based on harvest-time VNIR spectral models were also more accurate (± 0.38%) than the equivalent harvest-time SSC predictions. In addition, harvest-time DM predictions were shown to be capable of at least rank ordering (R2 = 0.87) kiwifruit in terms of post-storage SSC.

CHAPTER 3: INFLUENCE OF STORAGE DURATION ON PHYSICO- CHEMICAL CHANGES IN FRUIT OF APPLE CULTIVARS

Ibadullah Jan and Abdur Rab Department of Horticulture, Khyber Pakhtunkhwa Agricultural University Peshawar, Pakistan

Abstract The experiment on “Influence of storage duration on physico-chemical changes in fruit of apple cultivars” was conducted at Department of Horticulture, Khyber Pakhtunkhwa Agricultural University Peshawar, Pakistan during the years 2007-08. The fruit of apple cultivars: Royal Gala, Mondial Gala, Golden Delicious and Red Delicious were harvested at optimum maturity and stored at 5±1°C with 60-70% relative humidity. Physico-chemical changes in fruit were determined at 30 days interval during storage. Apple cultivar Red Delicious had the highest juice content (58.47%), TSS/Acid ratio (23.12), ascorbic acid (13.12 mg/100g), fruit flesh firmness (5.98 kg/cm2), fruit density (0.82 g/cm3) as well as the least weight loss (2.22%) but also had the highest bitter pit (11.86%) and soft rot (13.53%) incidence. Titratable acidity was the highest (0.55%) in cultivar Mondial Gala and starch score was the maximum (5.22) in cultivar Golden Delicious. The percent weight loss, total soluble solids, total sugar, pH, TSS/Acid ratio, bitter pit incidence and soft rot increased with increase in storage duration while juice content, starch score, titratable acidity, ascorbic acid, fruit flesh firmness and density of fruit declined with increase in storage duration.

3.1. INTRODUCTION

Apple (Pyrus domestica L) is one of the most important tree fruit of the world. The apple was cultivated in Greece around 600 BC or earlier. It is a highly nutritive fruit which is a rich source of sugars 11%, fat 0.4%, protein 0.3%, carbohydrates 14.9%, vitamins and minerals. A 100 g fresh apple contains, water 84.7 %, fibre 0.8 g, carbohydrates 13.9 g, proteins 0.4 g, lipid 0.3 g, ash 0.3 g, vitamin C 8 mg/100 gm, sodium 0.3 mg/100 g, potassium 145 mg/100 g, calcium 7 mg/100 g, magnesium 6 mg/100 gm, iron 480 μg/100 g, phosphorus 12 mg and iodine 2 μg (Hussain, 2001). Due to its high nutritional value, it ranks third in consumption after citrus and banana (Bokhari, 2002). In Pakistan its cultivation is limited and restricted to the northern hilly tracks of Punjab and Khyber Pakhtunkhwa, and the Quetta region of Balochistan. In Khyber Pakhtunkhwa, the apple plantation is distributed in Swat, Dir, Mansehra, Parachinar, Chitral, Hunza, North and South Waziristan Agencies. District Swat, with an area of approximately 4000 square miles with in the Malakand Division, is the most important of all the apple producing districts of Khyber Pakhtunkhwa followed by the districts of Mansehra, Dir, Abbottabad, Chitral and Hunza (Bokhari, 2002; Ali et al., 2004). The extended cultivation of apple in low altitude areas of Pakistan is limited by its high chilling requirements (Janick, 1974). Due to its chilling requirements, it grows best in relatively cooler climates than other deciduous fruits (Westwood and Chestnut, 1964). Apples can endure quite low temperatures, but temperatures of –30°C and rapid fluctuation in winter from relatively warm to extremely cold temperatures are harmful (Bokhari, 2002). The apple gives better yield in relatively long, cool and slow growing season, the type of climate which usually prevails at altitudes of 1700-2500 m. Apple trees grow well in a wide range of soil types. They prefer soils with a texture of sandy loam to a sandy clay loam. Good soil drainage is also critical for successful apple production. Ideal soil pH for apple tree is near 6.5. (Gao, 2001). The growth and development of apple tree is adversely affected by water logged soils, rising water tables into the root zone even for a short time, soils having hardpan and shallow soils (Chaudhary, 1994). Currently, the apple is grown over an area of 11.13 thousand hectares with a total production of 437.39 thousand tons in Pakistan (MINFA, 2008- 09). Various cultivars of apples which are being grown in Pakistan include Top Red, Red Spur, Red Delicious, Golden Delicious, Super Gold, Red Chief, Apple Elite,

Stark Crimson, Oregon Spur, Red Rom Beauty, Royal Gala, Mondial Gala, Spartan and Double Red (Chaudhary, 1994). The apple cultivars grown in Pakistan may vary considerably in physico-chemical characteristics such as titratable acidity, soluble solids, fruit flesh firmness, ethylene production and weight loss in cold storage (Golias et al., 2008) which may in turn influence the texture and storage performance of apple cultivars (Perring, 1989; Hoehn et al., 2003). Being in high demand throughout the year, the apple is generally stored. In relatively cold climates, simple warehouses may be effective for short term storage (Mitropoulos and Lambrinos, 2000) but cold storage is required for long term storage and quality retention. It is estimated that about 17% of apples produced in Balochistan are lost during postharvest operations (Shah et al., 2002). In Pakistan, apples kept under the conditions of cold storage for 22 weeks losses were found to be 28 percent (Ilyas et al., 2007). Postharvest losses also depend on production area and market distance and the time of transportation. It is reported that the total losses in the apples transported from Quetta, Swat and Murree to Faisalabad market during the months of August, September and November were found to be 23, 20 and 25% respectively (Ilyas et al., 2007). The problem is further complicated by the fact that various cultivars may vary significantly in their storage performance (Golias et al., 2008). The postharvest losses may depend on external and internal conditions. Among the external conditions, temperature and relative humidity during postharvest handling operations are the most important factors influencing the storage performance of apple (LeBlanc et al., 1996), which affect the fruit flesh firmness, juice content, weight loss, pH, soluble solids content (SSC), and other quality parameters (Tu et al., 2000). The internal factors may vary due to differences in fruit physiology and anatomy. Saleh et al., (2009) reported that fruits of apple cultivars Golden Delicious, Starking Delicious, Star Cremson and Gala stored at 85-90% relative humidity and 0ºC for 0 to 180 days exhibited significant differences in physiological and anatomical parameters. The differences in storage performance may be due to ethylene production, responsible for the changes in texture and fruit flesh firmness and fruit softening (Knee et al., 1983; Larrigaudiere et al., 1997; Stow et al., 2000; Johnston et al., 2001; Nilsson and Gustavsson, 2007). Water loss may also vary significantly among apple cultivars resulting in significantly different weight loss even under similar storage conditions (Khan and Ahmad, 2005).

The physical properties of apple fruit may also have significant influence on storage performance of apple because it influences water loss (Karathanos et al., 1995), gas exchange (Ho et al., 2010) and subsequent storage life (Meisami et al., 2009). Physical properties such as density of fruit and juice and porosity can be used to determine internal quality of produce (Wilson and Lindsay, 1969; Hudson, 1975; Zaltzman et al., 1987). Fruit density has also been used as maturity index in many fruits and vegetables such as apricots, strawberries, tomato, pea, etc (Wolfe et al., 1974; Zaltzman et al., 1987). According to Zaltzman et al. (1987), the fruit density is also related to the content of juice and dry matter (Jordan et al., 2000) and may indicate the maturity as well as quality changes in fruit during storage (Mitropoulos and Lambrinos, 2000). The present experiment was, therefore, conducted to evaluate the influence of storage duration on physico-chemical changes in fruit of apple cultivars.

Objectives: 1. Evaluate the quality of different apple cultivars 2. Evaluate the storage performance of apple cultivars 3. Examine the changes in internal quality characteristics during storage of apple cultivars

3.2. MATERIALS AND METHODS

The research on “influence of storage duration on physico-chemical changes in fruit of apple cultivars” was carried out at Horticulture Postharvest Laboratory, Khyber Pakhtunkhwa Agricultural University, Peshawar-Pakistan during 2007-08. The apple cultivars: Royal Gala, Mondial Gala, Golden Delicious and Red Delicious were harvested at commercial maturity stage at Matta, Swat, located at 35° North Latitude and 72° and 30° East Longitude in Pakistan. For this purpose (4×6×3=72) seventy two healthy trees of uniform size and vigour were selected. The unhealthy, diseased and bruised fruits were discarded while fruits of uniform size were selected for the study. The fruits of each cultivar were divided into six groups each containing 30 fruits, packed in corrugated boxes and stored in cold storage for 0, 30, 60, 90, 120 and 150 days. At the end of each storage interval the fruits were brought to Khyber Pakhtunkhwa Agricultural University Peshawar, for physico-chemical studies. The experiment was laid out in completely randomized design (CRD) with twenty four treatment combinations repeated three times. The detail of the post-harvest experiment is as under Cultivars (V) = (Royal Gala, Mondial Gala, Golden Delicious and Red Delicious) Storage duration (S) = (0, 30, 60, 90, 120 and 150days) The data were recorded and statistically analyzed for the following post harvest quality parameters at 30 days intervals.

Weight loss (%) Five fruits in each treatment were separated for weight loss test. The initial weight of each fruit was noted with the help of electronic balance. The average loss of weight in all the treatments was calculated at 30 days intervals. The weight loss (%) was calculated as under: Initial weight - final weight Fruit weight loss (%) =  100 Initial weight

Percent juice content Juice was extracted from five randomly selected fruit from each treatment with the help of juice extracting machine, weighed and the percentage was computed as described by Rehman et al. (1982). Weight of juice fruit -1 Percent Juice = × 100 Average weight of fruit Starch content Starch content was calculated at 30 days intervals of 150 days storage with the help of starch-iodine test. Iodine solution was prepared by dissolving 6 g of KI in 400 ml of water, and then added 1 g of I2. Slices of fruit dipped into iodine solution for 1 minute. Then the slices removed from the solution and let stand for 2 minutes. Each slice washed quickly in water and estimated the percentage of starch. Starch showed up as dark blue area and white areas represented sugar. The starch content was calculated according to Generic chart scores (1-8), where 1 represents the least and 8 the highest starch scores.

Total soluble solids Total Soluble Solids of the fruit was determined at 30 days intervals of 150 days storage accordingly. Total soluble solids (TSS) were measured with a hand refractometer (Kernco, Instruments Co. Texas). The juice from sample fruits were thoroughly mixed and drop of juice was placed on the slab of brix refractometer and covered with a transparent led. The rotation was observed through the eye piece of the equipment.

Total sugars Reducing and non-reducing sugars was determined by the method as described in A.O.A.C (1990).

Reducing sugars (%) Reagents

Fehling-A: 34.65g of CuSO4. 5H2O was dissolved in 500ml distilled water. Fehling-B: 173 g sodium potassium tartarate and 50g of NaOH was taken in beaker.

About 100 ml of H2O was added and dissolved the chemicals by striking. The

solution was transferred to 500 ml flask and volume was made up to the mark with distilled H2O. Indicator: Methylene blue: The methylene blue of 0.2 grams was taken in a 100 ml volumetric flask and after dissolving in about 50 ml distilled H2O , the volume was made up to the mark by adding more distilled water. Procedure Ten ml of sample was taken in 100ml volumetric flask and volume was made with distilled water up to the mark. The burette was filled with this solution. Then 5ml of Fehling-A and 5ml of Fehling-B solution along with 0.10 ml distilled water was taken in a conical flask. The flask was heated till boiling without disturbing the flask. Sample solution was added from the burette drop by drop while boiling till the colour became brick red in the flask. A drop of methylene blue will be added as indicator in the boiling solution without shaking the flask. If colour changes from red to blue for a moment, reduction was not complete and then more juice solution was added till red colour persists. Calculations 5ml of Fehling A + 5ml of Fehling B = Xml of 10% syrup solution = 0.05g of reducing sugar. 100ml of 10% sample solution contain

0.05 × 100 = Yg of reducing sugar X ml Y × 100 Percent of reducing sugar in sample = 10

Non reducing Sugar (%) Procedure Ten ml of sample was taken in a 100ml volumetric flask and the volume was made up to the mark with distilled water 0.20 ml of this solution was taken in a flask and 10 ml of IN HCI will be added, and then heated this solution for 5-10 minutes. After cooling 10 ml of IN NaOH was added and made this solution up to 250 ml. This sample solution was taken in a burette. Then 5ml Fehling A and 5 ml Fehling B solution along with 10 ml of distilled water was taken in a conical flask and boiled. When boiling started, it was titrated against the sample solution from the burette till

changed to red brick colour. It was tested with methylene blue as indicator till red colour persisted. Calculations: X ml of syrup solution contains = 0.05g of reducing sugar. 250 × 0.05 50 ml of syrup solution contain = = y gm of reducing sugars X ml This 250ml of syrup solution was prepared from 20ml of 10% sample solution contain Y × 100/20 = Pg reducing sugar 10 ml of sample solution contain Pg of reducing sugar 100 ml of sample solution contain = P × l00/10 = Qg of total reducing sugar. Qg of reducing sugar = inverted sugar + free reducing sugar. Non reducing sugar = total reducing sugar = free reducing sugar. Percent acidity Acidity was determined by neutralization reaction (AOAC, 1990) Principle The sample of unknown acidity is titrated with a standard 0.1N NaOH solution. The completion of the reaction is visualized by using phenolphthalein as indicator and light pink colour showed the end point. The acidity is calculated as following. N × T × 0.0067 × 10 Acidity (%) = × 100 D × S Where, N = Normality of NaOH T = ml of 0.1 N NaOH used D = ml of sample taken for dilution S = ml of diluted sample taken for titration and 0.0067 is the constant factor

Fruit pH The fruit pH of all treatments in each replication during storage at 30 days interval was determined with the help of electronic pH meter.

TSS/Acid Ratio The total soluble solids and acid ratio was calculated with the help of following formula. Total soluble solids TSS/Acid = Titratable acidity Ascorbic acid (mg/ml) Ascorbic acid was determined by the standard method as reported in AOAC (1990). Dye solution for Ascorbic acid determination: Fifty mg of 2, 6 dichlorophenol indophenols dye and 42 mg of sodium bicarbonate was weighed, dissolved in hot distilled water and volume was made up to 250 ml. Fifty mg of standard ascorbic acid was taken in 50ml volumetric flask and the volume was made up 0.4% oxalic acid. This standard ascorbic acid was titrated against dye. Titration of the sample Ten ml of sample was taken in 100 ml volumetric flask and volume was made by adding 0.4% oxalic acid .Then 10 ml of prepared sample was taken in the flask and was titrated against dye until light pink colour appeared, which persisted for 15 seconds. Three consecutive readings were taken for each sample. The ascorbic acid was calculated by using the following formula; F  T  10 Ascorbic acid =  100 D  S (ml of ascorbic acid ) F = Factor from standardization = ml of dye T= ml of dye used for sample S= ml of diluted sample taken for titration. D= ml of sample taken for dilution

Fruit flesh firmness (kg/cm2) Data pertaining to fruit flesh firmness was recorded with the help of penetrometer (Effigi, 11mm Prob.) for five fruits per treatment (Pocharski, et al., 2000).

Density of fruit (g/cm3) Density of fruit for each treatment in each replication was calculated by water displacement method (Meisami, et al., 2009). M Density of fruit = V Where, M is the mass of fruit and V is the volume of fruit.

Bitter pit (%) Percent bitter pit incidence was observed visually in each treatment by calculating the surface area of each fruit covered with the symptoms of bitter pit at time 0 and 30 days interval of cold storage.

Soft rot (%) Percent soft rot in each replication of treatments was examined visually and counted during 150 days storage and their disease percentage of fruits was calculated by formula as under Number of diseased fruits Percent disease incidence (%) =  100 Total number of fruit

Statistical Procedures The data calculated on different parameters were subjected to Analysis of Variance (ANOVA) technique to observe the differences between the different treatment as well as their interactions. In cases where the differences were significant, the means were further assessed for differences through Least Significant Difference (LSD) test. Statistical computer software, MSTATC (Michigan State University, USA), was applied for computing both the ANOVA and LSD.

3.3. RESULTS

Weight loss (%) The data regarding percent weight loss revealed that there were significant variations among cultivars and storage duration but not their interaction (Table 3.1). The data presented in Table 3.1 indicates that there were significant differences in weight loss of different apple cultivars. The maximum weight loss (2.91%), recorded in cultivar Golden Delicious, followed by Royal Gala and Mondial Gala with 2.43 and 2.40% respectively. The difference in weight loss in these three cultivars was, however, non significant. The minimum weight loss of 2.22% was recorded in Red Delicious. The percent weight loss increased significantly with incremental increase in storage duration (R2 = 0.985), so that it increased from the minimum of 0% for fresh harvested fruits to maximum of 4.05 and 4.53% with 120 and 150 days respectively. The difference in weight loss between 120 and 150 days was, however, non significant.

Juice content (%) It is clear from Table 3.1 that percent juice content was significantly varied among cultivars and storage durations but their interaction was non significant. The data regarding juice content revealed significant variation among apple cultivars. The maximum juice content (58.47%) recorded in Red Delicious, while the minimum (51.34%) in Royal Gala followed by Golden Delicious and Mondial Gala with 51.45 and 53.38% respectively. However, the juice percentage was non significant among these cultivars (Table 3.1). A gradual decrease was observed in juice content with increasing storage duration. The juice content significantly decreased from 64.18% in fresh fruit to 43.32% in fruit stored for 150 days. The gradual increase from 4.45% between 0 and 30 days to 5.13% between 90 and 120 days was recorded in the rate of decrease but it was 3.28% between 120 and 150 days.

Loss of starch content (Score) It is clear from the data that there was significant variation in starch content among apple cultivars and storage durations while, the interaction of cultivars and storage durations was non significant (Table 3.1).

The starch content varied significantly among cultivars with the maximum starch content score (5.22) recorded in Golden Delicious, that was statistically at par with Red Delicious and Royal Gala. Mondial Gala, however, had significantly lower starch content score of 4.58. The loss of starch content significantly increased with incremental increase in storage duration. The maximum mean starch score (7.05) recorded in fresh fruit, decreased with increase in storage duration to the minimum score (2.54) at the end of 150 days storage at 5±1oC.

Total soluble solids The data presented in Table 3.1 regarding percent total soluble solids revealed that there was non significant variation among cultivars of apple. The storage durations significantly affected total soluble solids of juice but interaction effect was non significant. The storage durations significantly affected the percent total soluble solids of apples. A gradual increase was observed in total soluble solids with increasing the storage durations (R2 = 0.986). The maximum total soluble solids (13.08%) were recorded in fruits stored for 150 days as compared to the minimum total soluble solids (9.93%) observed in fresh harvested fruits.

Total sugar (%) The data presented in Table 3.1 indicated non significant differences in total sugar among apple cultivars. The storage duration significantly affected the total sugar but the interaction of cultivars × storage durations was non significant. A significant increase in total sugar recorded with increasing the storage duration. The maximum total sugar (12.33%) observed in fruit stored for 150 days as compared to minimum (9.58%) recorded for fresh fruit. The differences in total sugar observed between fruit stored for 30 and 60 days as well as 90 and 120 days were non significant.

Table 3.1. The effect of storage duration on weight lost (%), percent juice (%), starch, TSS (%) and total sugar (%) of apple cultivars

Weight Juice Starch TSS Total Cultivar loss (%) content (%) (Score) (%) sugar (%) Royal Gala 2.43 ab 51.34 b 4.86 ab 11.36 10.81 Mondial Gala 2.40 ab 53.38 ab 4.58 b 11.64 10.93 Golden Delicious 2.91a 51.45 b 5.22 a 11.68 10.79 Red Delicious 2.22 b 58.47 a 4.94 ab 12.03 10.98 LSD at α 0.05 0.43 3.12 0.51 ns ns Storage Duration (days) 0 0.00 64.18 a 7.05 a 9.93 e 9.58 d 30 1.06 d 59.73 b 6.22 b 10.73 d 10.05 c 60 2.06 c 56.43 bc 5.68 c 11.39 cd 10.75 c 90 3.25 b 51.73 c 4.74 d 12.25 bc 10.98 b 120 4.05 a 46.60 d 3.18 e 12.70 ab 11.60 b 150 4.53 a 43.32 d 2.54 f 13.08 a 12.33 a LSD at α 0.05 0.43 3.08 0.67 0.69 0.60 Interactions Significance Level C × S ns ns ns ns ns

Mean followed by similar letter(s) in column do not differ significantly ns = Non Significant * = Significant at 5% level of probability. C × S = Interaction of cultivar and storage duration

Titratable acidity (%) Perusal of the data indicated significant variations in titratable acidity among different apple cultivars and storage durations but the interaction of cultivars × storage durations was non significant (Table 3.2). The data presented in Table 3.2 indicates that there were significant differences in percent acidity of different apple cultivars. The maximum titratable acidity (0.55%) was recorded in cultivar Mondial Gala, followed by Royal Gala and Golden Delicious with 0.54 and 0.51%. The difference in titratable acidity in these three cultivars was, however, non significant. The minimum titratable acidity (0.48%) recorded in Red Delicious. A significant decrease was observed in titratable acidity of apple juice with increasing storage duration (R2 = 0.989). The maximum titratable acidity (0.67%), observed in fresh harvested fruits while the minimum (0.38%) was recorded for fruits stored for 150 days.

Fruit pH The data regarding pH in Table 3.2 indicated non significant variation among apple cultivars; however, the effect of storage durations was significant. The interaction was non significant. A gradual increase in pH was recorded with increase in storage duration. The maximum pH (4.23) observed for fruits stored for 150 days while minimum pH (3.52) recorded for fresh harvested fruits. The effect of storage duration on pH of apple fruits was non significant for fruits stored up to 90 days and was also non significant for fruits stored between 120 and 150 days.

TSS/Acid ratio The data pertaining to TSS/Acid ratio revealed that there were significant differences among apple cultivars and storage durations, however, their interaction was non significant (Table 3.2). The maximum TSS/Acid ratio (26.81) recorded in Red Delicious was followed by Golden Delicious with 24.38. The minimum TSS/Acid ratio (22.18) recorded in Royal Gala, followed by Mondial Gala with 22.95, however these two cultivars at par with each other. Storage duration had a significant effect on TSS/Acid ratio of apple juice. The TSS/Acid ratio gradually increased with the increase in storage duration. The TSS/Acid ratio increased from 15.00 in freshly harvested fruit to 34.95 in apple fruit stored for 150 days. Ascorbic acid (mg/100g)

There were significant variation in ascorbic acid among apple cultivars, storage durations and cultivars × storage durations (Table 3.2). The data presented in Table 3.2 indicates that there were significant differences in ascorbic acid of different apple cultivars. The maximum ascorbic acid (13.12 mg/100g) recorded in cultivar Red Delicious followed by Royal Gala and Mondial Gala with 11.30 and 11.03 mg/100g, the difference in later two cultivars was, however, non significant. The minimum ascorbic acid (9.82 mg/100g) recorded in Golden Delicious. The ascorbic acid decreased significantly with incremental increase in storage duration (R2 = 0.994) so that it decreased from the maximum of 14.12 mg/100g observed with 0 day storage to minimum of 10.27 and 8.87 mg/100g with 120 and 150 days storage respectively. The interaction effect of cultivars and storage duration was also significant. The maximum ascorbic acid (15.60 mg/100g) was recorded with 0 days storage in Red Delicious while the minimum (7.22 mg/100g) was observed with 150 days storage in cultivars Golden Delicious. The percent decline in ascorbic acid in different cultivars over 150 days storage was the maximum 42.95% in cultivars Royal Gala followed by Mondial Gala and Golden Delicious with 41.26 and 38.13% respectively. While, cultivar Red Delicious not only had the maximum ascorbic acid at 0 day storage, it was also characterized by relatively slow decline during storage (27.12%) (Figure 3.1).

Table 3.2. The effect of storage duration on percent titratable acidity, pH, TSS/Acid ratio and ascorbic acid (mg/100g) of apple cultivars

Titratable Ascorbic acid Cultivar acidity (%) pH TSS/Acid ratio (mg/100g) Royal Gala 0.54 a 3.80 22.18 b 11.30 b Mondial Gala 0.55 a 3.75 22.95 b 11.03 b Golden Delicious 0.51 a 3.92 24.38 ab 9.82 c Red Delicious 0.48 b 3.79 26.81 a 13.12 a LSD at α 0.05 0.05 ns 2.71 0.70 Storage Duration (days) 0 0.67 a 3.52 b 15.00 e 14.12 a 30 0.62 a 3.58 b 17.57 e 13.06 b 60 0.57 b 3.70 b 20.28 d 11.06 c 90 0.48 c 3.78 b 25.78 c 10.53 cd 120 0.42 d 4.09 a 30.91 b 10.27 d 150 0.38 e 4.23 a 34.95 a 8.87 e LSD at α 0.05 0.05 0.34 2.68 0.67 Interactions Significance Level C × S ns ns ns *

Mean followed by similar letter(s) in column do not differ significantly ns = Non Significant * = Significant at 5 % level of probability. C × S = Interaction of cultivar and storage duration

Figure 3.1 Interaction effect of storage duration and cultivar on ascorbic acid (mg/100g) of apple fruit

Physical Quality Attributes of Apple Cultivars

Fruit flesh firmness (kg/cm2) It is clear from the Table 3.3 that fruit flesh firmness significantly varied among cultivars and storage durations, however, the interaction effect was non significant. The fruit flesh firmness significantly varied among apple cultivars. The maximum fruit flesh firmness (5.98 kg/cm2) was observed in Red Delicious, while the minimum fruit flesh firmness (5.15 kg/cm2) was recorded for Mondial Gala followed by Royal Gala and Golden Delicious with 5.19 and 5.27 kg/cm2 respectively, however, the effect was non significant among these cultivars. The fruit flesh firmness significantly decreased with increase in storage duration (R2 = 0.988). It decreased from the maximum of 6.59 kg/cm2 for fresh fruits to the minimum of 4.04 kg/cm2 for fruits stored for 150 days (Table 3.3).

Density of Fruit (g/cm3) The difference in density of apple fruit in relation to different cultivars and storage duration was significant, whereas, its interaction effect was non significant (Table 3.3). The maximum density of fruit (0.82 g/cm3) recorded in Red Delicious, followed by Golden Delicious and Mondial Gala with density of 0.80 and 0.78 g/cm3 respectively. However, the difference was non significant among these three cultivars. The minimum density of fruit (0.77 g/cm3) was recorded in Royal Gala. A significant decrease in density of fruit was recorded with increase in storage duration. The density of fruit decreased from the maximum of 0.82 g/cm3 recorded in fresh harvested fruit to the minimum of 0.77 g/cm3 after 150 days storage. However, the effect of storage duration was non significant on density for fruits stored up to 120 days.

Bitter pit (%) There was significant variation in percent bitter pit among different apple cultivars, storage durations and the interaction of cultivars × storage durations (Table 3.3). The apple cultivars varied significantly in bitter pit incidence of apple fruits. The maximum bitter pit incidence (11.86%) recorded in Red Delicious, was followed by Golden Delicious with 9.33% of bitter pit incidence. The bitter pit incidence in Royal

Gala and Mondial Gala was 5.02 and 5.90% respectively, with the difference being non significant. The bitter bit incidence gradually increased with incremental increase in storage durations (R2 = 0.971). The bitter bit increased from 0% for fresh harvested fruit to maximum of 18.85% for fruit after 150 days storage. The bitter pit incidence increased significantly with increasing storage duration in all the cultivars under study. After 150 days storage, the bitter pit incidence was significantly lower in Royal Gala and Mondial Gala with 10.51 and 11.57% respectively with the difference being non significant. The maximum bitter pit incidence of 29.63% recorded in cultivar Red Delicious (Figure 3.2).

Soft rot (%) The data in Table 3.3 revealed that the cultivars and storage durations as well as its interaction significantly influenced the percent soft rot incidence on apple fruits. Significant differences among apple cultivars were observed for percent soft rot during storage. The soft rot was maximum (13.53%) for Red Delicious, whereas, minimum percent soft rot incidence (8.82%) was observed in Mondial Gala, followed by Royal Gala and Golden Delicious with 8.93 and 10.56% respectively. The soft rot of apple fruit increased with incremental increase in storage durations. The soft rot (0%) gradually increased to maximum of 24.98% after 150 days storage. The interaction of cultivars and storage duration showed significantly high soft rot incidence with increasing storage duration. After 150 days storage, the soft rot incidence was the maximum (30.50%) in cultivars Red Delicious followed by Golden Delicious and Mondial Gala with 25.30 and 22.80%, with these two cultivars statistically at par with each other. The lowest soft rot incidence (21.30%) was observed in cultivar Royal Gala with the same storage duration (Figure 3.3).

Table 3.3. The effect of storage duration on fruit flesh firmness (kg/cm2), density of fruit (g/cm3), soft rot (%) and bitter pit (%) of apple cultivars

Cultivar Fruit flesh Density of Bitter pit Soft rot firmness Fruit (%) (%) (kg/cm2) (g/cm3)

Royal Gala 5.19 b 0.77 b 5.02 c 8.93 bc Mondial Gala 5.15 b 0.78 ab 5.90 c 8.82 c Golden Delicious 5.27 b 0.80 ab 9.33 b 10.56 b Red Delicious 5.98 a 0.82 a 11.86 a 13.53 a LSD at α 0.05 0.25 0.05 1.01 1.17 Storage Duration (days) 0 6.59 a 0.82 a 0.00 0.00 30 6.31 b 0.81ab 2.06 e 3.20 e 60 5.78 c 0.80 ab 5.45 d 6.45 d 90 5.18 d 0.78 ab 8.23 c 11.63 c 120 4.50 e 0.78 ab 13.58 b 16.50 b 150 4.04 f 0.77 b 18.85 a 24.98 a LSD at α 0.05 0.25 0.05 1.00 1.68 Interactions Significance level C × S ns ns * *

Mean followed by similar letter(s) in column do not differ significantly ns = Non Significant * = Significant at 5 % level of probability. C × S = Interaction of cultivar and storage duration

Figure 3.2 Interaction effect of storage duration and cultivar on bitter pit incidence (%) of apple fruit

Figure 3.3 Interaction effect of storage duration and cultivar on soft rot (%) of apple fruit 3.4. DISCUSSION

Weight loss (%) and Juice content (%) Moisture content of fruits is a major quality criteria (Gorini et al., 1979; Hatfield and Knee, 1988) and its loss from the fruits is serious consideration. Moisture loss decreases the visual quality and contributes to the loss of turgor pressure and subsequent softening (Vander-Beng, 1981). While the loss of moisture contributes to weight loss, yet it may not depend on the water content. This suggestion is strengthened by the observation that that Red Delicious had the highest juice content but significantly lower weight loss (Table 1.1). It suggests that weight loss is not simply a function of water present in the fruit. The structure of the skin and nature of waxes on the surface of the fruit may be important regulators of moisture loss in apples (Babos et al., 1984; Veravrbeke et al., 2003). Considerable variation has been observed in the skin thickness of different apple cultivars and even the same cultivar may show significant variation with in different years of production (Homutova and Blazek, 2006). The least weight loss in Red Delicious may be due to thicker waxy layer, characteristics of this cultivar (Veraverbeke et al., 2001). The moisture and subsequent weight loss in fruits increased linearly with increase in storage duration (R2 = 0.985) due water loss and respiration (Blampired, 1981; El-Shennawi, 1989; Erturk, 2003; Gavlheiro et al., 2003; Ghafir et al., 2009). The juice content of apple fruit depends on the water content of the fruit and the rate of water loss and hence can be decreased by increasing relative humidity (Tu et al., 2000). Since cultivar Red Delicious had the minimum weight loss, thus, it is likely to have high juice percentage. Similarly, cultivars exhibiting more weight loss were less juicy (Dzonova et al., 1970). The decrease in percent juice decline is due to the water loss from the tissue which increases with storage duration (Allan et al., 2003).

Loss of starch content (Score) The starch content of apple fruit depends mainly on the cultivars which may show significant variation (Ghafir et al., 2009). The storage durations significantly affected the starch content of apple. Since starch is the major storage carbohydrates in apple fruit (Beaudry et al., 1989), it is converted to sugars at the onset of ripening and during storage to meet the respiratory demand of the fruit (Bidabe et al., 1970; Crouch, 2003).

Total soluble solids Total soluble solids of apple and other fruits is a major quality parameter which is correlated with the texture and composition (Weibel et al., 2004; Peck et al., 2006). Ali et al., (2004) reported significant variations in TSS, acidity and other physico- chemical characteristics of apples harvested from different varieties but the different cultivars under study exhibited non significant variations in total soluble solids. The total soluble solids increased during storage (Mahajan, 1994; Rivera, 2005). The increase in TSS could be attributed to the breakdown of starch (Beaudry et al., 1989) into sugars (Bidabe et al., 1970; Crouch, 2003) or the hydrolysis of cell wall polysaccharides (Ben and Gaweda, 1985).

Total sugar (%) The sugars content, sucrose, glucose, fructose, and sorbitol, in fruit flesh contribute to the fruit sweetness, and is one of the major characteristics of fruit quality and market value. The apple fruit accumulate starch at the early stages of maturation that is later on hydrolyzed to sugars at edible maturity (Magein and Leurquin, 2000). The starch to sugars conversion continue during storage (Beaudry et al., 1989), resulting in increased total sugars with storage duration (Bidabe et al., 1970; Crouch, 2003). The increase in sugars during storage is therefore in line with the observation on loss of starch during the storage period.

Titratable acidity (%) and Fruit pH The data presented in Table 3.2 indicates that there were significant differences in percent acidity of different apple cultivars. The maximum titratable acidity recorded in cultivar Mondial Gala followed by Royal Gala and Golden Delicious while the lowest titratable acidity was in Red Delicious and it declined with the increase in storage duration. The changes in titratable acidity are significantly affected by the rate of metabolism (Murata and Minamide, 1970; Clarke et al., 2001) especially respiration which consumed organic acid and thus decline acidity (Rivera, 2005; Ghafir et al., 2009). While there was no significant difference in pH among apple cultivars however, the pH declined gradually with increasing storage durations. The pH of the fruit depends mainly on organic acid in the fruit, which are consumed in respiration, resulting lower acidity and high pH with increasing storage duration (Khalid, 1974; Chang et al., 1999; Rivera, 2005; Ghafir et al., 2009).

TSS/Acid ratio Total soluble solids of apple and other fruits is a major quality parameter (Weibel et al., 2004; Peck et al., 2006). Apple cultivars have been shown to have significant differences in TSS and acidity (Ali et al., 2004). The TSS/Acid ratio was significantly different among apple cultivars. Cultivar Red Delicious had the highest TSS/Acid ratio followed by Golden Delicious and Royal Gala while Mondial Gala had the lowest TSS/Acid ratio. The TSS/Acid ratio in all cultivars increased with increasing storage duration. The increase in TSS/Acid ratio can be attributed to starch breakdown resulting in free sugars (Beaudry et al., 1989) and decline in organic acids due to its consumption in respiration (Mahajan, 1994; Rivera, 2005; Ghafir et al., 2009).

Ascorbic acid (mg/100g) Ascorbic acid is usually considered as an index of nutrient quality in apple fruit. Ascorbic acid is a bioactive compound having antioxidant properties (Lata, 2007). While, the peel of apple fruit is generally a rich source of Vitamin C, but is correlated with the flesh Vitamin C content (Boyer and Liu, 2004). The apple cultivars differ significantly in their ascorbic acid content (Davey et al., 2007). For example apple cultivars Mushhadi and Amri had significantly higher vitamin C than Kalakulu (Ali et al., 2004; Nour et al., 2010) but generally is about 12.8 mg/100 g fruit (Lee et al., 2003). In the different apple cultivars under study, Red Delicious had the highest Ascorbic acid followed by Royal Gala and Mondial Gala while it was the lowest in Golden Delicious. The ascorbic acid decreased significantly with incremental increase in storage duration (Purvis, 1983; Hayat et al., 2003). The ascorbic acid in fruits and vegetables is sensitive to storage temperature or duration (Adisa, 1986) and its degradation is enhanced by adverse handling and storage conditions such as higher temperatures, low relative humidity, physical damage, and chilling injury. Beside abiotic factors, the ascorbic acid can be irreversibly oxidized (Parviainen and Nyyssonen, 1992; (Pardio-Sedas et al., 1994), which decreases the edible quality and increases susceptibility to different physiological disorders during storage (Jung and Watkins, 2008). The percent decline in ascorbic acid of different cultivars over 150 days storage revealed that the maximum ascorbic acid (42.95%) observed in cultivars Royal Gala followed by Mondial Gala and Golden Delicious with 41.26 and 38.13%

respectively. While, cultivar Red Delicious not only had the maximum ascorbic acid at 0 days storage, it was also characterized by relatively slow decline during storage (27.12%). The apple cultivars may vary not only in their initial ascorbic acid content (Ali et al., 2004; Nour et al., 2010) but also the rate of decline during storage.

Physical Quality Attributes of Apple Cultivars

Fruit flesh firmness (kg/cm2) Fruit flesh firmness is an important criteria for edible quality and market value of apples (Stow, 1995; De-Ell et al., 2001) and loss of fruit flesh firmness is a serious problems resulting in quality losses (Kov et al., 2005). The apple cultivars varied significantly in fruit flesh firmness with Red Delicious having the maximum while Mondial Gala had the minimum fruit flesh firmness. The fruit flesh firmness of the fruit depends on rate of evapo-transpiration, respiration rates, resulting in loss of solutes and water (Blampired, 1981; El-Shennawi, 1989; Gavlheiro et al., 2003; Erturk, 2003; Ghafir et al., 2009). The optimum fruit flesh firmness and texture of apple fruit, is a major quality parameter (Weibel et al., 2004; Peck et al., 2006). The difference in fruit flesh firmness of different apple cultivars may be their pectin composition (Billy et al., 2008), who reported that Golden Delicious softened easily during storage as compared to „‟ apples. We observed cultivar Golden Delicious had more fruit flesh firmness as compared to Royal Gala, Mondial Gala but was inferior to Red Delicious. The fruit flesh firmness of the apple fruit significantly decreased with increasing storage duration (Table 1.3). The fruit flesh firmness of the apple fruit is due to texture of the flesh and textural changes of fruits during ripening due to disassembly of primary cell wall and middle lamella structures (Jackman and Stanley, 1995; Cosgrove et al., 1997) and results in soft and mealy fruit that is less desired by consumers (Gomez, et al., 1998). The post harvest softening of apple fruit is believed to be related to cell wall breakdown (Fuller, 2008) due to enzymatic activities (Yamaki and Matsuda, 1977) and pectin solubalization (Bartley et al., 1982; Jackman and Stanley, 1995; Chang-Hai et al., 2006), thus reducing the mechanical strength of cell walls which decrease the fruit flesh firmness in apple fruits (Kov and Felf, 2003; Kov et al., 2005).

Density of Fruit (g/cm3) The density of the fruit is a physical characteristic that can be used to determine the quality of certain products. Potato‟s tuber‟s density is generally correlated with the starch content of tubers (Zaltzman et al., 1987), dry matter (Wilson and Lindsay, 1969), as well as the mechanical resistance of tubers (Hudson, 1975). The density of the fruit may be influenced by rind thickness (Cohen, 1972), juice content (Zaltzman et al., 1987), dry matter (Jordan et al., 2000) total sugars and starch contents (Robert et al., 2000). Thus, fruit density also been used as maturity and quality index in many fruits and vegetables such as apricots, strawberries, tomato, pea, etc (Wolfe et al., 1974; Zaltzman et al., 1987; McGlone et al., 2007). The apple cultivars varied significantly in fruit density (Vincent, 1989) which could be due to differences in biochemical composition (Homutova and Blazek, 2006; Ghafir et al., 2009) and moisture loss during storage (Rivera, 2005). Since the apple fruit lose considerable moisture during storage, its density tends to decrease during storage. The changes in density of apple fruit is a function of air spaces and solutes dissolved in the cell sap (Marlow and Loescher, 1984; Bayindirli, 1993). Therefore, the specific gravity is high in fresh fruits and decline during storage (Sakiyama and S. Nakamura, 1976) due to collapse of intercellular spaces and loss of moisture (Mitropoulos and Lambrinos, 2000).

Bitter pit (%) Bitter pit is a physiological disorder that appears as depressed brown lesions in the skin of the fruit, located mainly on the calyx end (Ferguson and Watkins, 1989). The apple cultivars varied significantly in bitter pit incidence with Red Delicious being the most susceptible followed by Golden Delicious. The significant variation among cultivars is in agreement with the findings of Crouch, (2003), who reported that apple cultivar Red Delicious is more susceptible to bitter pit as compared to Golden Delicious. The incidence of bitter pit depends on genetic factors as well as growth conditions and maturity at harvest (Crouch, 2003) or Ca concentration of the fruit (Pesis et al., 2009) and other nutrition (Fallahi et al., 1997). The incidence of bitter pit increased with increasing storage duration (Pesis et al., 2009). While the symptoms of bitter pit were not visible immediately after harvest but 18.85% of the fruit showed its symptoms after 150 days storage. The incidence of bitter pit depends on cultivars as well as growth conditions and maturity at harvest (Crouch, 2003; Pesis et al., 2009).

The lower bitter pit incidence in Royal Gala and Mondial Gala indicates the resistance of these cultivars to bitter pit incidence (Spotts et al., 1999). However, the high Bitter pit incidence in Red Delicious, despite high ascorbic acid, suggests that its incidence may not be related ascorbic acid content of the fruit as suggested by Mattheis and Rudell (2008) (Figure 1.2).

Soft rot (%) It is found that some cultivars are more susceptible to decay than the others. Thus the incidence of different pathogens on apple fruit such as Blue Mold, Gray Mold, Bull‟s- eye rot, and Mucor rot is dependent on cultivar (Spotts et al., 1999). While there was no soft rot incidence after harvest, significant differences among apple cultivars were observed after storage for 150 days with the maximum soft rot in Red Delicious while it was the lowest in Mondial Gala (Table 1.3). The apple cultivars are generally selected for resistance to certain postharvest diseases. For example, „Royal Gala‟ is extremely resistant to wound pathogens, „Granny smith‟ to skin punctures, and „Braeburn‟ to infiltration of fungal spores into the core (Spotts et al., 1999). While, the fruit may show considerable variation at early stages of maturation but the onset of ripening and senescence in various fruits and vegetables render them more susceptible to infection by pathogens (Kader, 1985). The increase in soft rot incidence with increasing storage duration varied significantly with cultivars. While cultivars Royal Gala had the least soft rot incidence, cultivars Red Delicious had the 30.16% high soft rot incidence than Royal Gala. It has previously been reported that Royal Gala‟ is extremely resistant to wound pathogens (Spotts et al., 1999) and hence had low soft rot incidence. Attempts have been made to correlate the loss of ascorbic acid (Davey et al., 2004) with increased susceptibility to physiological disorders and pathogens in apple fruit during storage (Lau, 1998; Watkins et al., 2003). It has been suggested that the development of physiological disorders in apple fruit during extended storage may be due to decreased antioxidant activities of different antioxidant compounds including ascorbic acid (Watkins et al., 2003), yet no correlation is observed between ascorbic acid and the incidence of soft rot or bitter pit (Mattheis and Rudell, 2008). But it is observed that Red Delicious, having higher ascorbic acid at any storage interval (Figure 1.3) but also high soft rot incidence. It suggests that soft rot incidence may not be correlated with ascorbic acid (Mattheis and

Rudell, 2008) but rather the general senescence and subsequent susceptibility of the fruit to decay (Kader, 1985).

3.5. SUMMARY, CONCLUSION AND RECOMMENDATIONS

Summary The research on “influence of storage duration on physico-chemical changes in fruit of apple cultivars” was carried out at Horticulture Postharvest Laboratory, Khyber Pakhtunkhwa Agricultural University Peshawar, Pakistan during 2007-08. The fruits were harvested from apple cultivars: Royal Gala, Mondial Gala, Golden Delicious and Red Delicious at commercial maturity stage at Matta, Swat and stored for 0, 30, 60, 90, 120 and150 days. The storage duration significantly affected the physico- chemical attributes of different apple cultivars during storage. Among the apple cultivars, Red Delicious showed better performance in regards to quality during storage. Red Delicious had the minimum weight loss (2.22%) and retained the maximum juice content (58.47%), TSS/Acid ratio (23.12), ascorbic acid (13.12 mg/100g), fruit flesh firmness (5.98 kg/cm2) and fruit density (0.82 g/cm3). On other hand, the cultivar Red Delicious was more susceptible to the incidence of bitter pit (11.86%) and soft rot (13.53 %) as compared to other cultivars. Red Delicious not only had the maximum ascorbic acid at 0 days storage, it was also characterized by relatively slow decline during storage (27.12%). Cultivar Golden Delicious has the lowest ascorbic acid and highest weight loss, thus measures should be taken to decrease weight loss during long term storage of this cultivar. The lowest fruit flesh firmness, ascorbic acid, starch content was recorded in cultivar Mondial Gala and also had the least soft rot incidence. Royal Gala has the lowest incidence of bitter pit but had poor quality characteristics during 150 days storage.

Conclusions o Apple cultivar Red Delicious is characterized by high juice content, TSS/Acid ratio, ascorbic acid, fruit flesh firmness and density and the least weight loss during storage. o The apple cultivar Red Delicious, despite good quality attributes, was found to be most susceptible to bitter and soft rot incidence, especially with prolong storage. Hence further study on decreasing the incidence of both the physiological disorders and soft rot is required to endure prolong storage.

o Cultivar Golden Delicious has the lowest ascorbic acid and highest weight loss, thus measures should be taken to decrease the rate of weight loss during long term storage of this cultivar. o Cultivar Mondial Gala being the lowest in firmness, ascorbic acid, starch content is relatively poor in quality but has the least soft rot incidence, while cultivar Royal Gala has the lowest incidence of bitter pit but has poor quality characteristics. Thus, both cultivars can be stored for longer duration than Golden Delicious or Red Delicious.

Recommendations  Apple cultivar Red Delicious due to its high juice content, TSS/Acid ratio, ascorbic acid, fruit flesh firmness and density and the least weight loss during storage can be recommended for refrigerated storage for 150 days.  The apple cultivar Red Delicious is more susceptible to bitter and soft rot incidence, especially with prolong storage. Hence precautionary treatments should be used to minimize the incidence of bitter pit and soft rot for extended storage duration e.g. 150 days.  Since cultivar Golden Delicious has the highest weight loss, thus high relative humidity in storage should be ensured to decrease the rate of weight loss during long term storage of this cultivar.  Cultivar Mondial Gala with the least soft rot incidence and cultivar Royal Gala with the lowest incidence of bitter are recommended for long term storage despite relatively poor quality attributes.

CHAPTER 4: STORAGE PERFORMANCE OF APPLE CULTIVARS HARVESTED AT DIFFERENT STAGES OF MATURITY

Ibadullah Jan and Abdur Rab Department of Horticulture, Khyber Pakhtunkhwa Agricultural University Peshawar, Pakistan

Abstract

An experiment was conducted to study “Storage performance of apple cultivars harvested at different stages of maturity” at Horticulture Postharvest Laboratory, Khyber Pakhtunkhwa Agricultural University Peshawar Pakistan during 2007-08. The fruits were harvested at three different stages of maturity at fifteen days interval representing early, mid and late harvesting stage from apple cultivars: Royal Gala, Mondial Gala, Golden Delicious and Red Delicious and evaluated for different quality parameters at 0 and 150 days storage at 5±1°C with 60-70% relative humidity. The maximum weight loss (2.83%) was recorded in cultivar Golden Delicious, whereas the minimum juice content (52.04%) in cultivar Royal Gala, which was at par with 52.43% juice content Golden Delicious, while starch score, TSS, total sugars were non significantly different among cultivars. Cultivar Red Delicious had the highest TSS/Acid ratio (26.73), ascorbic acid (12.49 mg/100g), fruit flesh firmness (5.85 kg/cm2) but also the bitter pit (14.22%) and soft rot (15.52%) incidence, while titratable acidity (0.56%) and fruit density (0.80 g/cm3) was observed in cultivar Mondial Gala as compared to the least titratable acidity (0.50%) was recorded in cultivar Red Delicious. The percent weight loss, total soluble solids, total sugar, pH, TSS/Acid ratio, bitter pit incidence and soft rot increased while, juice content, starch score, titratable acidity, ascorbic acid, fruit flesh firmness and density of fruit declined with increase in storage duration. The juice content (47.68%), total soluble solids (10.07), total sugar (9.31%), pH (3.71), TSS/Acid ratio (18.73), ascorbic acid (10.11 mg/100g) and soft rot (9.52%) recorded with early mature fruit, increased to juice content (59.33%), total soluble solids (12.92), total sugar (12.98%), pH (4.23), TSS/Acid ratio (29.29), ascorbic acid (12.50%) and soft rot (15.22%) accordingly in late mature fruits, while weight loss (3.34%), starch score (4.95), titratable acidity (0.59%), fruit flesh firmness (5.88 kg/cm2), density of fruit (0.82 g/cm3) and bitter pit (11.69%) recorded at early maturity stage, declined with delaying the harvesting to weight loss (1.93%), starch score (3.21), titratable acidity (0.49%), fruit flesh firmness (4.81 kg/cm2), density of fruit (4.81 g/cm3) and bitter pit (6.63%) at late maturity stage.

4.1. INTRODUCTION

Apple (Pyrus domestica L) is an important fruit desired for taste and nutritive value (Bokhari., 2002). Apple is cultivated in the northern and hilly areas of Pakistan (Ali et al., 2004; Bokhari., 2002) over an area of 11.13 thousand hectares with a total production of 437.39 thousand tons (MINFA, 2008-09). The apple fruit is in high demand throughout the year and hence a considerable quantity is generally stored in cold storages in Pakistan. Apple being a perishable commodity is prone to qualitative and quantitative losses after harvest. The losses may occur during postharvest operations or storage which could be as high as 17% (Shah et al., 2002) or even greater (Ilyas et al., 2007). The postharvest quality and losses in apple fruit may depend on cultivar (Saleh et al., 2009), cultural practices (Tomala, 1999), nutritional status (Raese et al., 1989), harvesting stage (Strief, 1996; Vielma et al., 2008) and storage condition (LeBlanc et al., 1996).

The storage life of apple can be increased and post harvest losses decreased by selecting the best adopted cultivars, provision of optimum nutrition and harvesting at optimum stage (Strief, 1996). In Pakistan, the apples are harvested at edible maturity for both fresh market and storage. Fruit harvested at this stage are more prone to mechanical injury, have short storage life and greater susceptibility to pathogens and physiological disorders (Hribar et al., 1996). In addition, careless harvesting characterized by immature and over mature fruit, is another serious cause of post harvest losses (Ingle et al., 2000). Being a climacteric fruit, the apple can be harvested at physiological maturity, stored and ripened artificially to catch good price in the market. In general, apple fruit harvested at immature stage have poor colour and flavour and can be more susceptible to physiological disorders such as bitter pit and superficial scald (Juan et al., 1999). By contrast, fruit harvested over-mature tend to be soft and easily damaged during post harvest operations (Hribar et al., 1996). Such fruits are more susceptible to diseases and physiological disorders as well as quality deterioration during or after storage (Lafer, 2006). A wide range of indices has been tested over many years as possible indicators of harvest maturity (Lau., 1985). Ethylene production and starch content have been commonly used to predict the maturity of apple fruit (Lau., 1985) but relationships between ethylene production and optimum harvest dates can be poor, and the timing of increased ethylene production is

a function of cultivar as well as growing region, orchard within a region, cultivar strain, growing season conditions, and nutrition (Watkins, 2003). The problem is further complicated by the fact that several different cultivars are grown in Pakistan which may vary considerably in their time of maturity and storage performance (Ozelkok et al., 1995; Drake et al., 2002). Differences in storage performance may be due to pre harvest conditions, biochemical composition (TSS and Soluble sugars) or physical characters such as fruit flesh firmness, surface wax etc. (Veravrbeke et al., 2003; Rizzolo et al., 2006; Vielma et al., 2008). According to Ferguson and Watkins (1989) the incidence and severity of bitter pit depend on cultivar as well as growth condition and time of harvest and considerable decrease in bitter pit and core flush can be achieved by harvesting at optimum stage and storage at proper conditions (Lau, 1992). For long term storage, fruit need to be harvested before they begin producing ethylene (Roth et al., 2005). While the genetic characteristics of the apple fruits for storage may vary, yet optimal storage may retain good organoleptic quality longer than suboptimum storage conditions (Sestras et al., 2006). Thus, it is essential that the optimum harvest stage in apple cultivars is identified for maximum fruit quality and storage life and minimize post harvest losses in apple (Beaudry et al., 1993; Blanpied and Silsby, 1992, Streif, 1996).

Objectives: 1. Examine the changes in internal quality during storage of apple cultivars 2. Determine the influence of harvesting stages on the quality and storage performance of apple cultivars

4.2. MATERIALS AND METHODS

The research on “Storage performance of apple cultivars harvested at different stages of maturity” was carried out at Horticulture Postharvest Laboratory, Khyber Pakhtunkhwa Agricultural University Peshawar, Pakistan during 2007-08. The fruits were harvested at three different stages of maturity at fifteen days interval representing early, mid and late harvesting stage from four apple cultivars: Royal Gala, Mondial Gala, Golden Delicious and Red Delicious. Healthy trees of uniform size and good vigour were selected for harvesting fruit samples. Fruit showing the symptoms of surface damage or abnormalities were discarded while fruits of uniform size were selected for the study. The fruits harvested at each stage from different cultivars were divided into two groups each containing 50 fruits. One lot was analyzed for different quality attributes while the other was shifted to cold storage and stored for 150 days (SD 150) at 5±1oC and 60-70% relative humidity. The calcium content of soil and leaves was determined by using the following procedures:

Preparation of Soil Extract by AB-DTPA Ten g of air dried soil sieved through 2 mm was taken and added 20 ml of AB-DTPA solution. The solution was shacked for 15 minutes and filtered through wattman 42 filter paper. The reading was taken through atomic absorption spectrophotometer.

Extraction of Plant Samples The sample was first grinded to a particle size of 1mm and 1 g of plant sample was taken in digestion flask. It was left over night after adding 10 ml HNO3. Added 4 ml perchloric acid (HClO4) and heated for 20 minutes and then solution become colourless. The sample was cooled and transferred to volumetric flask and the volume was made to the mark. This solution was used for recording the calcium content in leaves and fruits through atomic absorption spectrophotometer (Isaac and Kerber, 1971).

Table 4.1. Calcium content (mg/kg) of orchard soil at the time of fruit picking Harvesting stage Cultivar Early Mid Late Royal Gala 486 478 471 Mondial Gala 453 441 434 Golden Delicious 511 499 475 Red Delicious 425 413 399

Table 4.2. Calcium content (%) of apple leaves at the time of fruit picking Harvesting stage Cultivar Early Mid Late Royal Gala 1.25 1.38 1.43 Mondial Gala 1.46 1.56 1.62 Golden Delicious 1.20 1.37 1.58 Red Delicious 1.08 1.18 1.29

The experiment was laid out in three factor completely randomized design (CRD) having twenty four treatment combinations repeated three times. The detail of the post-harvest experiment is as under

Cultivars (V) = (Royal Gala, Mondial Gala, Golden Delicious and Red Delicious) Harvesting stages (H) = Early, Mid and Late Storage duration (S) = (0 and 150days)

The data was recorded and statistically analyzed for the following post harvest quality parameters as described in experiment No. 1 at the interval of 0 and 150 days of storage at 5±1oC. Weight loss (%), Percent juice content, Starch content, Total soluble solids (0Brix), Total sugars, Percent acidity, Fruit pH, TSS/Acid ratio, Ascorbic acid (mg/100g), Fruit flesh firmness (kg/cm2), Density of fruit, Bitter pit (%) and Soft rot (%)

Statistical Procedures The data calculated on different parameters were subjected to Analysis of Variance (ANOVA) technique to observe the differences between the different treatment as well as their interactions. In cases where the differences were significant, the means were further assessed for differences through Least Significant Difference (LSD) test. Statistical computer software, MSTATC (Michigan State University, USA), was applied for computing both the ANOVA and LSD.

4.3. RESULTS

Weight Loss (%) The data on percent weight loss indicated that there were significant variations among cultivars, storage duration and harvesting stages. The interaction of cultivar × storage, cultivars × harvesting stage, storage × harvesting stage and cultivar × storage × harvesting stage was also significant (Table 4.3). The data presented in Table 4.3 indicates that there were significant differences in weight loss of different apple cultivars. The maximum weight loss (2.83%) recorded in cultivar Golden Delicious which was significantly higher than Royal Gala (2.43%), Red Delicious (2.42%) and Mondial Gala (2.35%), with the difference in weight loss in the later three cultivars being non significant. The mean percent weight loss increased significantly with storage for 150 days to maximum of 5.02%. Harvesting stages also significantly affected the weight loss. The maximum weight loss (3.34%) recorded with early harvesting stage, which was significantly higher than both mid and late harvesting stages. The difference in mid and late harvesting stages with 2.26 and 1.93% was non significant. The interaction effect of cultivars and storage duration was also significant. The maximum weight loss (5.67%) recorded in cultivar Golden Delicious after 150 days storage while it was 4.85 and 4.70% in cultivars Royal Gala, Red Delicious and Modial gala respectively. The difference in the later three cultivars was, however, non significant (Figure 4.1). The interaction of cultivars and harvesting stages significantly affected percent weight loss. The maximum weight loss (3.73%) observed in cultivar Golden Delicious at early harvesting stage. The weight loss tends to decline with advancing harvesting stage so that the minimum weight loss (1.77%) recorded in Red Delicious at late harvesting stage (Figure 4.2). The interaction of storage durations and harvesting stages also significantly affected the weight loss. The maximum weight loss (6.67%) recorded at early harvesting stage after 150 days storage (Figure 4.3). The interaction effect of cultivars, storage durations and harvesting stages was also significant on weight loss. The maximum weight loss (7.45%) observed in cultivar Golden Delicious at fruits harvested at early stage with storage of 150 days (Figure 4.4).

Juice content (%) The data regarding percent juice content revealed that there were significant differences among cultivars, storage duration and harvesting stages, however, the interaction effect was non significant (Table 4.3). The apple cultivars varied significantly in juice content with the maximum juice content (58.54%) recorded in Red Delicious. However the minimum juice content (52.04%) recorded in Royal Gala but it was non significant with Golden Delicious and Mondial Gala with 52.43 and 54.40% respectively. The juice content significantly decreased from 61.28 % for fresh harvest fruits to 47.43% for fruits 150 days storage. The harvesting stages also significantly affected the percent juice content of apple cultivars. Juice content increased from the minimum of 47.68 to 56.05% and finally to the maximum of 59.33% with harvest at early to mid and late stages respectively. The difference in juice content of mid and late harvested fruit was, however, non significant.

Loss of starch content (Score) The data pertaining to starch score revealed that there were significant decline with storage and harvesting stages, however, the difference among cultivars and interaction effect was non significant (Table 4.3). The starch content score decreased significantly during storage from 6.42 for fresh harvested fruit to 1.99 for fruit stored for 150 days. Harvesting stages significantly affected the starch content with the maximum starch content score (4.95) recorded at early harvesting stage, that declined to 4.45 and 3.21 when fruits were harvested at mid or over nature stage respectively.

Total soluble solids The data regarding total soluble solids indicated that there were non significant differences among cultivars, whereas, the effect of storage and harvesting stages was significant. The interaction of storage × harvesting stages was also significant (Table 4.3). The total soluble solids were significantly increased during storage, it from 10.02% for fresh harvested fruit to 12.95% for 150 days storage. Harvesting stages significantly affected the total soluble solids. The minimum total soluble solids (10.07%) at early harvesting stage increased with harvesting at mid mature stage

(11.47%) and finally to the maximum of 12.92% at late harvesting stage. The interaction of storage durations and harvesting stages also significantly affected total soluble solids. The maximum total soluble solids (13.99%) recorded at late harvesting stage after 150 days storage while the minimum total soluble solids (8.28%) recorded at 0 day storage at early harvesting stage (Figure 4.5).

Total sugar (%) The data on total sugar revealed no significant differences among cultivars, whereas, the effect of harvesting stages and storage was significant. The interaction effect of storage × harvesting stages was also significant (Table 4.3). The total sugar increased during storage from 9.67% for fresh harvested fruit to 12.47% in fruit stored for 150 days. The total sugar increased significantly with delayed harvesting stages from the minimum total sugar (9.31%) at early harvesting stage, followed by mid and late harvesting stages with 10.89 and 12.98% respectively. The interaction of storage durations and harvesting stages also significantly affected total sugar. The maximum total sugar (14.04%) recorded at late harvesting stage after 150 days storage while the minimum total sugar (7.63%) observed at early harvesting stage after 0 day storage (Figure 4.6).

Table 4.3. The effect of harvesting stages and storage on weight lost (%), percent juice, starch, TSS (%) and total sugar (%) of apple cultivars

Cultivar Weight Juice Starch TSS Total Loss (%) Content (Score) (%) sugar (%) (%) Royal Gala 2.43 b 52.04 b 4.08 11.26 10.83 Mondial Gala 2.35 b 54.40 ab 4.08 11.24 10.85 Golden Delicious 2.83 a 52.43 b 4.17 11.66 11.29 Red Delicious 2.42 b 58.54 a 4.50 11.79 11.31 LSD at α 0.05 0.30 5.34 Ns ns ns Storage (days) 0 0.00 61.28 6.42 10.02 9.67 150 5.02 47.43 1.99 12.95 12.47 Significance level * * * * * Harvesting Stages Early 3.34 a 47.68 b 4.95 a 10.07 c 9.31 c Mid 2.26 b 56.05 a 4.45 a 11.47 b 10.89 b Late 1.93 b 59.33 a 3.21 b 12.92 a 12.98 a LSD at α 0.05 0.35 6.25 0.80 0.68 0.71 Interactions Significance Level C × S * ns Ns ns ns C × H * ns Ns ns ns S × H * ns Ns * * C × S × H * ns Ns ns ns

Mean followed by similar letter(s) in column do not differ significantly ns = Non Significant * = Significant at 5 % level of probability. C × S = Interaction of cultivar and storage duration, C × H = Interaction of cultivar and harvesting stage S × H = Interaction of storage duration and harvesting stage C × S × H = Interaction of cultivar, storage duration and harvesting stage

7

6

5

4

3

Weight Loss (%) Loss Weight 2

1

0 Royal gala Mondial gala Golden delicious Red delicious Cultivars

Figure 4.1 Variation in percent weight loss among apple cultivars after 150 days storage

4.5 4 3.5 3 Early 2.5 Mid 2 Late 1.5

Weight Loss (%) Loss Weight 1 0.5 0 Royal gala Mondial gala Golden Red delicious delicious Cultivars

Figure 4.2 Interaction effect of cultivar and harvesting stage on percent weight loss in apple fruit

8 7 6 5 4 3

2 Weight Loss (%) Loss Weight 1 0 Early Mid Late Harvesting stages

Figure 4.3 Influence of harvesting stages on percent weight loss in apple fruits after 150 days storage

9 8 7 6 Early 5 Mid 4 Late 3

2 Weight Loss (%) Loss Weight 1 0 Royal gala Mondial gala Golden Red delicious delicious Cultivars

Figure 4.4 Interaction effect of cultivar and harvesting stage on percent weight loss of apple fruits stored for 150 days

16 14 12 10 0 8 150 6 4

2 Total Soluble Solids (%) Solids Soluble Total 0 Early Mid Late Harvesting Stages

Figure 4.5 Interaction effect of storage and harvesting stage on total soluble solids of apple

16

14

12

10 0 8 150 6

Total Sugars (%) Sugars Total 4

2

0 Early Mid Late Harvesting Stages

Figure 4.6 Interaction effect of storage and harvesting stage on total sugar of apple

Titratable Acidity (%) The data in Table 4.4 indicated that there were significant differences in titratable acidity among cultivars, storage and harvesting stages. The interaction storage × harvesting stages significantly affected the titratable acidity, however, all other interactions were non significant. There were significant differences in percent acidity of different apple cultivars. The maximum titratable acidity (0.56%) recorded in cultivars Mondial Gala and Royal Gala, while minimum titratable acidity (0.50%) observed in Red Delicious, followed by Golden Delicious with 0.51% however, the difference was non significant. The percent acidity was significantly declined with the increase in storage that decreased from 0.69% observed in fresh harvested fruits to 0.37% for fruits stored for 150 days. A significant variation in titratable acidity was observed in relation to harvesting stages. The maximum titratable acidity (0.59%) recorded in the apple fruits at early harvesting stage. The minimum titratable acidity (0.49%) measured at late harvesting stage followed by mid harvested fruits with 0.52% however, the variation was non significant. The interaction of storage durations and harvesting stages also significantly affected titratable acidity. The maximum titratable acidity (0.79%) recorded at early harvesting stage after 0 days storage while the minimum titratable acidity (0.35%) observed at late harvesting stage after 150 days storage (Figure 4.7).

Fruit pH The data pertaining to fruit pH revealed that there were non significant differences among cultivars, whereas, the effect of storage and harvesting stages was significant. The interaction effect of cultivars and storage duration was also significant (Table 4.4). A significant increase in pH observed during storage, which increased from the minimum (3.54) for fresh harvested fruit to the maximum (4.27) for fruit stored for 150 days. Harvesting stages significantly affected the pH, the maximum pH (4.23) recorded at late harvesting stage, however, the minimum pH (3.71) observed at early harvesting stage, followed by mid harvesting stage with 3.90. The interaction effect of cultivars and storage duration was also significant. The maximum pH (4.48) recorded in cultivar Golden Delicious after 150 days storage, while the least pH (3.41) observed in cultivar Mondial Gala with 0 day storage (Figure 4.8).

TSS/Acid ratio The data regarding TSS/Acid ratio indicated significant variations among apple cultivars, storage and harvesting stages. The interaction effect was non significant (Table 4.4). TSS/Acid ratio was the maximum (26.73) for cultivar Red Delicious followed by Golden Delicious (24.63) with the difference being non significant. Significantly lower TSS/Acid ratios of 22.49 and 22.76 were observed in cultivars Mondial Gala and Royal Gala respectively which were statistically at par with each other. Storage had a significant effect on TSS/Acid ratio of apple juice. The TSS/Acid ratio increased from 16.88 in fresh fruit to 31.42 with storage for 150 days. The least TSS/Acid ratio (18.73) observed in early harvested fruits, increased non significantly to 24.43 with mid harvested fruits and then significantly to 29.29 when harvesting was done at late mature stage.

Ascorbic acid (mg/100g) The data on ascorbic acid as shown in Table 4.4, indicated that ascorbic acid content of apple fruit varied significantly with cultivars, storage and harvesting stages as well as the interaction of cultivar × storage and storage × harvesting stage. The maximum ascorbic acid content recorded in cultivar Red Delicious (12.49 mg/100g) followed by Mondial Gala and Royal Gala with 11.53 and 11.43 mg/100g respectively. The least ascorbic acid observed in cultivar Golden Delicious (10.27 mg/100g) (Table 4.4). The ascorbic acid decreased significantly from 14.18 to 8.68 mg/100g during 150 days storage at 5±1oC. Harvesting stages also significantly affected the ascorbic acid content of apple fruit. Ascorbic acid was the lowest in fruits harvested at early maturity stage (10.11 mg/100g), which increased to 11.68 and 12.50 mg/100g in fruits harvested at mid and over mature stages respectively. The interaction effect of cultivars and storage durations on ascorbic acid was also significant. The maximum ascorbic acid (14.76 mg/100g) recorded in cultivar Red Delicious after 0 day storage, while the minimum ascorbic acid (8.19 mg/100g) observed with 150 days storage in cultivar Royal Gala (Figure 4.9). The interaction of storage durations and harvesting stages also significantly affected ascorbic acid. The maximum ascorbic acid (15.23 mg/100g) recorded at late harvesting stage after 0 day storage while the minimum ascorbic acid (7.11 mg/100g) recorded at early harvesting stage after 150 days storage (Figure 4.10).

Table 4.4. The effect of harvesting stages and storage on percent titratable acidity, pH, TSS/Acid ratio and ascorbic acid (mg/100g) of apple cultivars Cultivar Titratable pH TSS/Acid Ascorbic acidity (%) ratio acid (mg/100g) Royal Gala 0.56 a 3.93 22.76 b 11.43 b Mondial Gala 0.56 a 3.67 22.49 b 11.53 b Golden Delicious 0.51 b 4.04 24.63 ab 10.27 c Red Delicious 0.50 b 3.97 26.73 a 12.49 a LSD at α 0.05 0.05 ns 3.28 0.64 Storage (days) 0 0.69 3.54 16.88 14.18 150 0.37 4.27 31.42 8.68 Significance level * * * * Harvesting Stages Early 0.59 a 3.71 b 18.73 c 10.11 c Mid 0.52 b 3.90 ab 24.43 b 11.68 b Late 0.49 b 4.23 a 29.29 a 12.50 a LSD at α 0.05 0.05 0.37 3.85 0.75 Interactions Significance Level C × S ns * ns * C × H ns ns ns ns S × H * ns ns * C × S × H ns ns ns ns

Mean followed by similar letter(s) in column do not differ significantly ns = Non Significant * = Significant at 5 % level of probability. C × S = Interaction of cultivar and storage duration, C × H = Interaction of cultivar and harvesting stage S × H = Interaction of storage duration and harvesting stage C × S × H = Interaction of cultivar, storage duration and harvesting stage

1 0.9 0.8 0.7 0.6 0 0.5 150 0.4 0.3

Titratable Acidity (%) Acidity Titratable 0.2 0.1 0 Early Mid Late Harvesing Stages

Figure 4.7 Interaction effect of storage and harvesting stage on titratable acidity (%) of apple

6

5

4 0

3 pH 150 2

1

0 Royal gala Mondial gala Golden Red delicious delicious Cultivars

Figure 4.8 Interaction effect of cultivar and storage on pH of apple fruits

18 16 14 12 10 0 8 150 6 4 2 Ascorbic Acid Acid (mg/100g) Ascorbic 0 Royal gala Mondial gala Golden Red delicious delicious Cultivars

Figure 4.9 Interaction effect of cultivar and storage ascorbic acid (mg/100g) of apple

18 16 14 12 10 0 8 6 150 4

2 Ascorbic Acid Acid (mg/100g) Ascorbic 0 Early Mid Late Harvesting Stages

Figure 4.10 Interaction effect of storage and harvesting stage on ascorbic acid (mg/100g) of apple

Fruit flesh firmness (kg/cm2) Significant differences in fruit flesh firmness of apple fruits were observed with apple cultivars, storage and harvesting stages. The interaction effect was non significant (Table 4.5). The fruit flesh firmness varied significantly among apple cultivars. The maximum fruit flesh firmness (5.85 kg/cm2) observed in Red Delicious. The minimum fruit flesh firmness (5.08 kg/cm2) recorded for Royal Gala followed by Mondial Gala and Golden Delicious with 5.09 and 5.34 kg/cm2 respectively, however, the effect was non significant among these cultivars. The fruit flesh firmness significantly decreased from 6.62 kg/cm2 for fresh fruits to 4.09 kg/cm2 for fruits stored for 150 days. Harvesting stages significantly affected the fruit flesh firmness of apple fruit. The maximum fruit flesh firmness (5.88 kg/cm2) recorded at early harvested fruit, followed by fruits at mid harvesting stage with 5.33 kg/cm2 and the least fruit flesh 2 firmness (4.81 kg/cm ) observed at late harvesting stage.

Density of Fruit (g/cm3) The data on fruit density as given in Table 4.5 indicated that fruit density of apple fruit varied significantly with cultivars, storage and harvesting stages, however, the interaction effect was non significant (Table 4.5). Fruit density of apple fruit significantly varied among cultivars. The maximum density of fruit (0.81 g/cm3) observed in Mondial Gala, followed by Red Delicious and Golden Delicious with density of 0.80 and 0.79 g/cm3 respectively, however, the variation was non significant among these three cultivars. The minimum density of fruit (0.75 g/cm3) recorded in Royal Gala. A significant decrease in density of fruit recorded during storage. The fruit density decreased from 0.81 g/cm3 for fresh harvested fruits to 0.77 g/cm3 recorded in fruits stored for 150 days. Harvesting stages significantly affected the apple fruit density. The fruits harvest at early stage had the highest density (0.82 g/cm3), followed by fruits harvested at mid and late stages of maturity with 0.79 and 0.76 g/cm3 respectively.

Bitter pit (%) The data on bitter pit incidence indicated that significant differences were observed in apple fruit with cultivars, storage and harvesting stages as well as the interaction of cultivar × storage and storage × harvesting stage (Table 4.5).

The apple cultivars significantly varied in relation to bitter pit incidence. The maximum bitter pit incidence (14.22%) observed in Red Delicious, followed by Golden Delicious with 11.23%. The minimum bitter pit (5.72%) recorded in Royal Gala, followed by Mondial Gala with 5.77%, with the difference being non significant. Bitter bit incidence significantly increased to 18.47% with 150 days storage. Harvesting stages had significantly affected the bitter pit incidence. The maximum bitter pit incidence (11.69%) observed in fruits at early harvesting stage, followed by mid and late harvesting stages with 9.39 and 6.63% respectively (Table 4.5). The interaction of cultivars and harvesting stages significantly affected percent bitter pit. The maximum bitter pit incidence (17.70%) observed in cultivar Red Delicious at early harvesting stage while the minimum bitter pit incidence (3.97%) recorded in Mondial Gala at late harvesting stage (Figure 4.11). The interaction of storage durations and harvesting stages also significantly affected the incidence of bitter pit. The maximum bitter pit incidence (23.38%) recorded at early harvesting stage after 150 days storage (Figure 4.12).

Soft rot (%) Soft rot of apple fruit varied significantly with cultivars, storage and harvesting stages as well as the interaction of cultivar × storage and storage × harvesting stage, however, all other interaction was non significant (Table 4.5). Significant differences among apple cultivars have been recorded for percent soft rot during storage. The soft rot was maximum (15.52%) for Red Delicious, whereas, minimum percent soft rot (10.53%) observed in cultivar Royal Gala, followed by Mondial Gala and Golden Delicious with 10.83 and 12.76% respectively. The soft rot in apple fruit significantly increased to 24.82% during 150 days storage. Soft rot significantly increased with delaying harvesting time. The maximum soft rot (15.22%) observed at late harvesting stage, followed by mid and early harvesting stage with 12.49 and 9.52% respectively (Table 4.5). The interaction of cultivars and harvesting stages significantly affected percent soft rot incidence. The maximum soft rot (19.45%) observed in cultivar Red Delicious at late harvesting stage while minimum soft rot (7.75%) recorded in Royal Gala at early harvesting stage (Figure 4.13). The interaction of storage durations and harvesting stages also significantly affected percent soft rot. The maximum soft rot (30.43%) recorded at late harvesting stage after 150 days storage (Figure 4.14).

Table 4.5. The effect of harvesting stages and storage on fruit flesh firmness (kg/cm2), fruit density (g/cm3), soft rot (%) and bitter pit (%) of apple cultivars

Cultivar Fruit flesh Density of Bitter pit Soft rot firmness fruit (g/cm3) (%) (%) (kg/cm2) Royal Gala 5.08 b 0.75 b 5.72 c 10.53 c Mondial Gala 5.09 b 0.81 a 5.77 c 10.83 bc Golden Delicious 5.34 b 0.79 ab 11.23 b 12.76 b Red Delicious 5.85 a 0.80 ab 14.22 a 15.52 a LSD at α 0.05 0.36 0.05 2.31 2.21 Storage (days) 0 6.62 0.81 0.00 0.00 150 4.09 0.77 18.47 24.82 Significance level * * * * Harvesting Stages Early 5.88 a 0.82 a 11.69 a 9.52 c Mid 5.33 b 0.79 ab 9.39 a 12.49 b Late 4.81 c 0.76 b 6.63 b 15.22 a LSD at α 0.05 0.42 0.05 2.71 2.59 Interactions Significance Level

C × S ns ns ns ns C × H ns ns * * S × H ns ns * * C × S × H ns ns ns ns

Mean followed by similar letter(s) in column do not differ significantly ns = Non Significant * = Significant at 5 % level of probability. C × S = Interaction of cultivar and storage duration, C × H = Interaction of cultivar and harvesting stage S × H = Interaction of storage duration and harvesting stage C × S × H = Interaction of cultivar, storage duration and harvesting stage

25

20

15 Early Mid

10 Late Bitter Pit (%) Pit Bitter 5

0 Royal gala Mondial gala Golden Red delicious delicious Cultivars

Figure 4.11 Interaction effect of cultivar and harvesting stage on bitter pit (%) of apple

30

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Bitter Pit (%) Pit Bitter 10

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0 Early Mid Late Harvesting Stages

Figure 4.12 Influence of harvesting stages on bitter pit (%) of apple fruits after 150 days storage

25

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15 Early Mid

10 Late Soft Rot (%) Rot Soft 5

0 Royal gala Mondial gala Golden Red delicious delicious Cultivars

Figure 4.13 Interaction effect of cultivar and harvesting stage on soft rot (%) of apple

40 35 30 25 20

15 Soft Rot (%) Rot Soft 10 5 0 Early Mid Late Harvesting Stages

Figure 4.14 Influence of harvesting stages on soft rot (%) of apple fruits stored for 150 days

4.4. DISCUSSIONS

Weight loss (%) Moisture loss from the fruits is serious consideration which decreases the visual quality and contributes to the loss of turgor pressure and subsequent softening (Vander-Beng, 1981). Apple cultivars vary significantly in weight loss (Veravrbeke et al., 2003). The mean weight loss was the highest in Golden Delicious followed by Royal Gala, Red Delicious and Mondial Gala. The rate of moisture from the fruit depends on skin thickness and nature of surface waxes (Babos et al., 1984; Veravrbeke et al., 2003), which may vary considerably in different apple cultivars or even in same cultivar in different years of production (Homutova and Blazek, 2006). Since, cultivar Red Delicious is characterized by a thicker waxy layer, (Veraverbeke et al., 2001), it may lose less moisture. Harvesting stages also significantly affected the weight loss. Generally, the weight loss in fruit harvested at early mature stages was significantly higher than both mid and late harvesting stages, while the difference in mid and late harvesting stages was non significant. Since, the weight loss in fruit depends on moisture loss (Bidabe, 1970; Ghafir et al., 2009), which is regulated by epi-cuticular waxes which increase with maturation (Lau, 1992). The high weight loss in fruit harvested at early stage of maturation may be due to poorly developed waxy surface and cuticle (Ihabi et al., 1998; Sass and Lakner, 1998). It may explain relatively lower weight loss in late harvested fruits that have fully developed waxy layer on their surface (Lau, 1992). The percent weight loss increased significantly with storage for 150 day. The moisture and subsequent weight loss in fruits generally increased with increase in storage duration (Tu et al., 2000). The loss of moisture from the fruit takes place through evapo-transpiration resulting in weight loss during storage (Bidabe, 1970; Ghafir et al., 2009). Thus, it is clear that the fruit lose weight due to moisture loss after harvest (Veravrbeke et al., 2003) and the loss moisture is more at early harvesting stage (Sass and Lakner, 1998) during storage (Tu et al., 2000), yet the rate of weight loss may vary significantly among different apple cultivars despite similar harvesting stage or storage duration (Veravrbeke et al., 2003).

Juice content (%) The juice content of apple fruit depends on water present in the fruit (Allan et al., 2003). The juice content of fruit was the maximum in cultivar Red Delicious but the minimum in Royal Gala. Since cultivar Red Delicious had the minimum weight loss, thus, it is likely to have high juice percentage. The mean juice content of the fruit also related to harvesting stages and tended to increase with delaying in harvesting late stages. The juice content of apple fruit depends mainly on the water content and its loss from the fruit. Thus, cultivars characterized by more weight loss are generally less juicy (Dzonova et al., 1970). This will explain the decrease in percent juice with increasing storage duration (Allan et al., 2003).

Loss of starch content (Score) While there was no significant difference in starch content of different cultivars under study, it decreased significantly with delaying harvesting from early mature stage to mid and late mature stages and storage for 150 days (Table 4.3). The starch is the major storage carbohydrates in apple fruit (Beaudry et al., 1989) and it is converted to sugars and water at the onset of ripening (Magein and Leurquin, 2000) and during storage (Beaudry et al., 1989) to meet the respiratory demand of the fruit (Bidabe et al., 1970; Marriot et al., 1981). Thus, the decrease in starch content with late mature harvesting stage or storage can be attributed to the conversion of starch to sugars (Crouch, 2003).

Total soluble solids Total soluble solids of apple and other fruits is a major quality parameter which is correlated with the texture and composition (Weibel et al., 2004; Peck et al., 2006). While Ali et al., (2004) reported significant variations in their TSS, acidity and other physico-chemical characteristics in apples harvested from different varieties but no significant variations were observed in total soluble solids of different cultivars in this study. The total soluble solids increased during storage for 150 days (Mahajan, 1994; Rivera, 2005). Since the starch content decline during storage (Table 4.3), it is likely to observe increased TSS (Bidabe et al., 1970; Beaudry et al., 1989; Crouch, 2003). Similarly, the hydrolysis of complex cell wall polysaccharides into simple sugars could be another pool responsible for increased TSS with storage (Ben and Gaweda, 1985). The interaction effect revealed that the percent increase in TSS was 30.51% in

fruits harvested at early stages, but 23.56 and 15.02% in fruits harvested at mid and later stages of harvest. It may due to more starch content in fruit harvested at early mature stages as compared to fruits harvested at mid or late mature stages (Table 4.3) and hence more conversion in early harvested fruits.

Total Sugar (%) The sugar content is one of the major characteristics of fruit quality and market value. Apple cultivars under study were statistically non significant for total sugars but significant differences were observed due to harvesting stages and storage duration. The minimum total sugars were recorded in fruits at early maturity stage which increased with delay to mid or late harvesting stage. Generally, there are significant variations in their TSS, acidity and other physico-chemical characteristics in apples harvested from different varieties (Ali et al., 2004) but no significant variations were observed in total soluble solids of different cultivars in this study. The total sugars increased during storage (Mahajan, 1994; Rivera, 2005) because of starch, hydrolyzed to sugars at edible maturity (Magein and Leurquin, 2000). The starch to sugars conversion continue even during storage (Beaudry et al., 1989), resulting increase in total sugars with storage duration (Bidabe et al., 1970; Crouch, 2003). Since the starch content decline during storage (Table 4.2), it is likely to observe increased TSS, predominantly sugars (Beaudry et al., 1989; Bidabe et al., 1970; Crouch, 2003). Similarly, the hydrolysis of complex cell wall polysaccharides into simple sugars could be another pool responsible for increased TSS with storage (Ben and Gaweda, 1985). The interaction effect revealed that the total sugars increased more in fruits harvested at early stages, but less in fruits harvested at mid and late maturity stages of harvest. It may due to more initial starch content in fruit harvested at early mature stages as compared to fruits harvested at mid or late mature stages (Table 4.3) resulting in high total sugars in early harvested fruits. The increased total sugar may in part be due to the loss of moisture during storage (Veravrbeke et al., 2003), resulting in higher concentration of sugars per unit volume of water. Thus, the increase in sugars during storage is therefore in line with the observation on loss of starch and moisture during the storage. Titratable acidity (%) and pH Significant differences in percent acidity were observed among different apple cultivars, Mondial Gala and Royal Gala having high titratable acidity than Red

Delicious or Golden Delicious. The titratable acidity of different cultivar, however, varied significantly with harvesting stages. The titratable acidity was the maximum in the apple fruits at early harvesting stage and then declined with delay in harvest to mid or late mature stage of harvest. A similar tendency was also observed in relation to storage, which resulted in significant declined in titratable acidity. The trend of pH was opposite to the trends in titratable acidity; with the maximum pH recorded at late harvesting stage as compared to lower pH as harvesting was done early mature stages. Similarly the pH in fresh fruit was higher than fruit stored for 150 days. The titratable acidity of the fruit depends on the rate of metabolism (Murata and Minamide, 1970; Clarke et al., 2001) especially respiration which consumed organic acid and thus decline acidity (Riveria, 2005). The fruit being living organs that respires even after harvested from the tree and during storage which consume the organic acids (Ghafir et al., 2009) and hence decrease the titratable acidity of the fruit (Ghafir et al., 2009). It results in a corresponding change in the pH of the fruit. (Chang et al., 1999). Since the fruit continue to respires even after harvested from the tree and during storage, the acidity decline and corresponding increase in pH is observed (Ghafir et al., 2009).

TSS/Acid ratio Total soluble solids, predominantly sugars, of apple and other fruits is a major quality parameter (Weibel et al., 2004; Peck et al., 2006). The balance between TSS and acidity is responsible for the taste of apple fruits (Hecke et al., 2006). Fruits with high TSS/Acid ratio are sweet while that of lower TSS/Acid ratio, are generally sour. Apple cultivars may have significant variation in TSS and acidity (Ali et al., 2004) but generally the TSS increase due to starch breakdown (Beaudry et al., 1989) while the acidity decline during storage (Mahajan, 1994; Rivera, 2005; Ghafir et al., 2009) due to its consumption in respiration (Riveria, 2005). Thus, it results in increased TSS/Acid ratio with increasing storage duration (Chang et al., 1999).

Ascorbic acid (mg/100g) Ascorbic acid is an important quality characteristic of apple fruit, specially desired for its antioxidant properties (Lata, 2007). Though, the peel of apple fruit is richer source of Vitamin C than the flesh, yet it is correlated with the flesh Vitamin C content (Boyer and Liu, 2004). The cultivars Red Delicious had the maximum ascorbic acid followed by Mondial Gala and Royal Gala while the least ascorbic acid observed in cultivar Golden Delicious. The apple cultivars differ significantly in their ascorbic

acid content (Ali et al., 2004; Nour et al., 2010). But it generally average around 12.8 mg/100 g fruit (Lee et al., 2003) and generally decline during storage (Purvis, 1983; Hayat et al., 2003). The ascorbic acid content of apple fruit may also depends on the stage at which the fruits are harvested. Generally, the fruit harvested at early maturity had lower ascorbic acid than later stages of harvest, indicating that the fruit may have still been synthesizing ascorbic acid when harvested at the early mature stages. Since the degradation of ascorbic acid is faster at higher than lower temperature (Pardio- Sedas et al., 1994), harvesting at late mature stage would have little ascorbic acid as compared to early harvested apples. Similarly, the early harvested apples tend to lose more ascorbic acid (45.81%) as compared to mid or late harvested fruit with 35.61 and 35.78% respectively. It is also interesting to note that while cultivars Royal Gala, Modial gala and Red Delicious had non significant differences in ascorbic acid content, the percent ascorbic acid loss of different cultivars revealed that Royal Gala lost 44.17% of its initial ascorbic acid as compared to only 30.69% in cultivar Red Delicious.

Fruit flesh firmness (kg/cm2) Fruit flesh firmness, that depends on total soluble solids (TSS) contents as well as the texture of apple fruit (Weibel et al., 2004; Peck et al., 2006). It is important for edible quality, postharvest handling and market value of apples (Stow, 1995; De-Ell et al., 2001). Thus, the loss of fruit flesh firmness during storage is a serious concern as it results in quality losses (Kov et al., 2005) leading to soft and mealy fruit and hence less consumers (Gomez, et al., 1998). The fruit flesh firmness varied significantly with cultivars, with Red Delicious being the most firm cultivar while Royal Gala was the least. The difference in fruit flesh firmness among apple cultivars indicate that apple cultivars may vary in their pectin composition and that causes rapid softening in Golden Delicious during storage (Billy et al., 2008). The fruit flesh firmness was also significant at different stages of maturity with the maximum fruit flesh firmness recorded in fruit harvested at early stages of maturity while the least observed in fruit harvested at late harvesting stage. The fruit flesh firmness of the fruit depends on the texture of the flesh and changes in primary cell wall during ripening (Cosgrove et al., 1997; Fuller, 2008). It may involve disassembly of primary cell wall and middle lamella structures (Jackman and Stanley, 1995) due to enzymatic activities (Yamaki and Matsuda, 1977) and pectin solubalization (Bartley et al., 1982; Jackman and

Stanley, 1995; Chang-Hai et al., 2006). Thus, the mechanical strength of cell walls is decreased with a concomitant decrease in the fruit flesh firmness of fruits (Kov et al., 2003; Kov et al., 2005).

Density of Fruit (g/cm3) The fruit density of apple significantly varied among cultivars with the highest density observed in Mondial Gala while the least in Royal Gala which decreased significantly during storage. Harvesting stages also significantly affected the apple fruit density where the fruit harvested at early maturity stage had the highest density followed by fruits harvested at mid and late stage of maturity. The density of apple fruit is a function air spaces and solutes dissolved in the cell sap (Esau, 1977). The juice content (Zaltzman et al., 1987), dry matter (Jordan et al., 2000), total sugars and starch contents (Robert et al., 2000) are important factors influencing fruit density. It has been used to determine the maturity and quality in different fruits and vegetables such as such as apricots, strawberries, tomato, pea, etc (Wolfe et al., 1974; Zaltzman et al., 1987; McGlone et al., 2007). The apple cultivars differ significantly in their density (Vincent, 1989) and larger fruit of the same cultivar may have a higher proportion of air spaces than smaller fruits (Volz et al., 2004) and hence have low density. While Modial Gala had the highest and Royal Gala had the least density, cultivars Red Delicious, Golden Delicious and Mondial Gala were not significantly different. Fruit with lower densities and more air volume per fruit have been shown to be softer (Volz et al., 2004), or more mealy (Tu et al., 1996) and more prone to physiological disorders (Marlow and Loescher, 1984). In accordance with the previous reports that the intercellular air spaces in apple fruit continues to increase during storage (Harker et al., 1997), the density of apple fruit decreased by 4.94% during storage for 150 days. As the cells expand during fruit growth, the volume of intercellular air spaces also increase and a considerable portion is occupied by it as the fruit approach maturity (Yamaki and Ino, 1992), which leads to a decline in fruit density, though the density of the fruit cells sap may change little (Westwood et al., 1967). The fruit density, thus, declined by 10.59% from early to late maturity stage of harvest.

Bitter pit (%) The sensitivity to bitter pit depends on genetic factors as well as growth conditions and maturity at harvest (Crouch, 2003; Pesis et al., 2009), thus it is likely to observe significant variations in different apple cultivars. It is observed that cultivars Red Delicious was more susceptible than Golden Delicious (Crouch, 2003) and while cultivar Royal Gala was the least sensitive to bitter pit incidence. Generally, the incidence of bitter pit significantly increases with increasing storage duration (Pesis et al., 2009) may be due to redistribution of calcium resulting in decreased calcium levels in the peel with increasing storage duration. Harvesting stages also significantly affected the bitter pit incidence so that it decreased by 43.28% in fruit harvested at late mature stage as compared fruit harvested at early mature stage. The apple fruits should be harvested at optimum maturity (Ingle et al., 2000) and both early and late harvest is not desirable (Meresz et al., 1993) with early harvested fruits being more sensitive to bitter pit (Juan et al., 1999). The interaction of cultivars and harvesting stages revealed that in all the cultivars under study, the bitter pit incidence percentage was the highest for early harvested fruit and declined with delaying harvesting to mid or late mature stages (Figure 4.11) (Ferguson et al., 1993).

Soft rot (%) The soft rot of apple fruit is due the development of brown and watery lesions on the skin of the apple that generally extend into the flesh (Watkins and Rosenberger, 2002). Significant differences among apple cultivars were observed among different cultivars with Red Delicious being the most sensitive (Watkins and Rosenberger, 2002.) than Royal Gala and Mondial Gala. Since apple cultivars are generally selected for resistance to certain postharvest diseases (Spotts et al., 1999), the incidence of different pathogens on apple fruit depend on cultivar (Spotts et al., 1999). The mean soft rot incidence significantly increased by 24.82% during 150 days storage and the increase was more in fruits harvested at later stage of maturity than fruits harvested at mid or early maturity stages (Kader, 1985). The interaction of cultivars and harvesting stages significantly affected percent soft rot incidence with the maximum soft rot observed in cultivar Red Delicious at late harvesting stage while minimum soft rot was recorded in Royal Gala at early harvesting stage (Figure 4.13). The soft rot incidence was the minimum at early mature stage of harvest but increased with advance in maturity stages (Erkan and Pekmezcu, 2004). It is a common observation

that the fruits which are more susceptible to different pathogens as they advance in ripening (Robertson et al., 1990) due to either senescence (Murray et al., 1998) or more susceptibility to mechanical injury (Kader, 2002).

4.5. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary The experiment on “Storage performance of apple cultivars harvested at different stages of maturity” was carried out at Horticulture Postharvest Laboratory, Khyber Pakhtunkhwa Agricultural University Peshawar, Pakistan during 2007-08. The fruits were harvested from four apple cultivars: Royal Gala, Mondial Gala, Golden Delicious and Red Delicious at three different stages of maturity at fifteen days interval representing early, mid and late harvesting stage. The storage performance of apple cultivars was significantly affected by the harvesting stages The highest juice content (58.54%), TSS/Acid ratio (26.73), ascorbic acid (12.49 mg/100g), fruit flesh firmness (5.85 kg/cm2) were recorded in cultivar Red Delicious but it showed the more susceptibility to bitter pit (14.22%) and soft rot (15.52%) as compared to other cultivars. The higher juice content, total soluble solids, total sugar, pH, TSS/Acid ratio, ascorbic acid, density of fruit as well as the minimum percent weight loss and bitter pit incidence were observed in the late harvested fruits as compared to early or mid harvested fruits. The fruit flesh firmness decreased (4.81 kg/cm2) with delaying harvesting below the acceptable range. The fruit harvested at late stage of maturity had high soft rot incidence during storage.

Conclusions o Apple cultivar Red Delicious has high juice content, TSS/Acid ratio, ascorbic acid and fruit flesh firmness. o Apple cultivar Red Delicious having high quality attributes but also has the highest bitter pit incidence, thus measures should be taken to minimize it during storage. o The apple cultivar Golden Delicious has the highest weight loss during long storage. It should be decreased by taking suitable measures during storage. o Cultivar Mondial Gala has the maximum titratable acidity and fruit density but the low fruit flesh firmness. o The apple cultivar Royal Gala has the lowest bitter pit incidence but all other quality attributes were poor.

o Early harvested fruits, despite low incidence of soft rot, were found to have the highest weight loss, bitter pit as well as the least juice content, TSS, total sugar and TSS/Acid ratio. o Fruits harvested at mid harvesting stage having relatively good quality parameters as compared to early harvested fruits and also the soft rot incidence was less as compared to late harvested fruits, thus can be stored for 150 days. o Late harvesting stage having good quality attributes but the fruits were very soft, had least starch content and also had the highest soft rot incidence.

Recommendations  Apple cultivar Red Delicious had superior quality due to its high juice content, TSS/Acid ratio, ascorbic acid and fruit flesh firmness.  Although, cultivar Red Delicious has high quality attributes but also has the highest bitter pit incidence. Thus, it can be recommended for short term storage i.e. less than 150 days storage.  The apple cultivar Golden Delicious has the highest weight loss during long storage and relatively high bitter pit and soft rot incidence. So, this cultivar cannot be recommended for extended storage i.e. 150 days.  Cultivar Mondial Gala and Royal Gala have relatively poor quality attributes but more resistant to bitter pit and soft rot incidence. Thus, these cultivars can be recommended for prolong storage.  Early harvested fruits, despite low incidence of soft rot, were found to have the highest weight loss and bitter pit also have the least juice content, TSS, total sugar and TSS/Acid ratio. Thus, early harvested fruits should not be stored for longer duration.  Fruits harvested at mid harvesting stage with relatively low incidence of bitter pit and soft rot as well as more fruit flesh firmness and high starch score are recommended for long term storage despite relatively poor quality attributes.  Late harvesting fruits have good quality attributes but very soft, less starch content and the highest soft rot incidence, thus late harvested fruits cannot be recommended for long term storage.

CHAPTER 5: INFLUENCE OF CaCl2 CONCENTRATION AND DIPPING DURATION ON INTERNAL QUALITY CHANGES IN APPLE CULTIVAR ‘RED DELICIOUS’

Ibadullah Jan and Abdur Rab Department of Horticulture, Khyber Pakhtunkhwa Agricultural University Peshawar, Pakistan

Abstract

The research on “Influence of CaCl2 concentration and dipping duration on internal quality changes in apple cv Red Delicious” was carried out at Horticulture Postharvest Laboratory, Khyber Pakhtunkhwa Agricultural University, Pakistan during 2009-10. The fruits were harvested at commercial maturity stage from apple cultivar “Red

Delicious” at Matta, Swat. The fruits were dipped in 0, 3, 6 and 9% CaCl2 solution for the period of 3, 6, 9 and 12 minutes and stored for 150 days at 5±1°C with 60-70% relative humidity. The percent weight loss, total soluble solids, total sugar, TSS/Acid ratio, bitter pit incidence and soft rot increased while, juice content, starch score, titratable acidity, ascorbic acid, fruit flesh firmness and density of fruit declined with increase in storage duration. The juice content (60.63%), starch score (5.05), ascorbic acid (12.67 mg/100g), fruit flesh firmness (5.98 kg/cm2) and density of fruit (0.81 mg/100g) recorded in fruits dipped in 0% CaCl2 solution (control), increase to juice content (64.34%), starch score (5.68), ascorbic acid (13.87 mg/100g), fruit flesh firmness (6.54 kg/cm2) and density of fruit (0.84 mg/100g) in fruits dipped in 9%

CaCl2 solution, whereas weight loss (1.95%), total soluble solids (12.01), total sugar (10.95%), TSS/Acid ratio (28.09), bitter pit incidence (15.18%) and soft rot (15.33%) incidence recorded in control, reduced with increase in CaCl2 concentration to weight loss (1.31%), total soluble solids (11.88), total sugar (10.76%), TSS/Acid ratio (23.88), bitter pit incidence (3.80%) and soft rot (2.10%) in fruits treated with 9% 2 CaCl2 solution. The starch score (5.30), fruit flesh firmness (6.13 kg/cm ) and density of fruit (0.82 mg/100g) recorded in fruits dipped for 3 minutes in CaCl2 solution, increase to starch score (5.50), fruit flesh firmness (6.49 kg/cm2) and density of fruit

(0.84 mg/100g) in fruits dipped for 12 minutes in CaCl2 solution, whereas weight loss (1.81%), TSS/Acid ratio (27.21), bitter pit incidence (11.92%) and soft rot (7.07%) incidence recorded in fruits dipped for 3 minutes in CaCl2 solution, reduced with increase in dipping duration to weight loss (1.41%), TSS/Acid ratio (25.27), bitter pit incidence (5.60%) and soft rot (6.28%) in fruits treated CaCl2 solution for 12 minutes.

5.1. INTRODUCTION

Apple (Pyrus domestica) has high nutritional value and it ranks third in consumption after citrus and banana (Bokhari, 2002). In Khyber Pakhtunkhwa, it is cultivated in Swat, Dir, Mansehra, Parachinar, Chitral, Hunza, North and South Waziristan Agencies. District Swat, is the most important of all the apple producing districts of Khyber Pakhtunkhwa followed by the districts of Mansehra, Dir, Abbottabad, Chitral and Hunza (Bokhari, 2002; Ali et al., 2004). The extended cultivation of apple in low altitude areas is limited by its high chilling requirements (Janick, 1974). The apple fruit is generally stored in cold storage because its demand is high through out the year. Apple is prone to qualitative and quantitative losses after harvest due to its perishability (Shah et al., 2002). The storage life is limited by loss of fruit flesh firmness (Kov et al., 2005), loss of chemical quality (Golias et al., 2008), physiological disorders (Juan et al., 1999) and disease incidence or decay (Ingle et al., 2000; Hribar et al., 1996). Thus, attempts have been made to explore different methods to decreases the postharvest losses during storage (Lau, 1992; Conway et al., 2002; Mahmud et al., 2008; Gupta and Jawandha, 2010). Calcium is an important second category macro-nutrient which is involved in regulating the metabolism in apple fruit, and adequate concentration maintains fruit flesh firmness, delays fruit ripening, lower the incidence of physiological disorders such as water core, bitter pit, and internal breakdown (Faust and Shear., 1968; Bangerth, et al., 1972; Mason, et al., 1975; Reid and Padfield., 1975) and suppress Erwinia carotovora (Jones) incidence on apple fruits (Sharples and Johnson., 1977; Conway, 1982). The fruit may experience Ca deficiency despite high Ca content in most orchard soils or Ca uptake by apple trees (Petersen, 1980) which may lead to several physiological disorders. Generally the apple fruits containing less than 50 mg kg−1 Ca of fresh weight are sensitive to bitter pit and internal breakdown (Petersen, 1980). By contrast adequate calcium helps to maintain apple fruit flesh firmness and decreases the incidence of physiological disorders such as water core, bitter pit and internal breakdown and postharvest decay (Conway et al., 2002). Soil treatments with calcium to increase fruit calcium concentration have often met with very little success but direct application of calcium to the fruit is the most effective method for increasing fruit calcium content, accomplished by pre-harvest sprays or postharvest dips or vacuum or pressure infiltration (Conway et al., 2002).

According to Martin et al. (1960), spraying trees of apples with magnesium nitrate increased the incidence of pit but calcium nitrate decreased it. Kadir (2005) reported that five to eight CaCl2 applications to „Jonathan' apple at fruits sizes of 0.9 and 1.6 cm average diameters retained fruit flesh firmness (26%) and the SSC/TA (35%) but fewer CaCl2 applications were required to sustain fruit skin colour during storage. Due to the repeated used of calcium spays in pre-harvest application, dipping in calcium solution or vacuum infiltration is commonly used to slow down the rate of softening during storage (Omaima et al., 2007).

Objectives:

1. To evaluate the influence of CaCl2 application on the quality and storage performance of apple fruit.

2. To optimize the CaCl2 concentration and dipping duration to minimize the quality losses during storage in apple cultivar Red Delicious.

5.2. MATERIALS AND METHODS

The influence of CaCl2 concentration and dipping duration on internal quality changes in apple cultivar „Red Delicious‟ was investigated at Postharvest Laboratory, Khyber Pakhtunkhwa Agricultural University, Peshawar-Pakistan during 2009-10. The fruits from apple cultivar Red Delicious were harvested at commercial maturity stage. The fruit were harvested from three different plants. The harvested fruits were checked from mechanical injury or symptoms of diseases and physiological disorders. Healthy and sound fruits of uniform size were selected and dipped in 0, 3, 6 and 9%

CaCl2 solution for 3, 6, 9 and 12 minutes. Calcium solution was made from analytical grade Calcium chloride. The fruit understudy were carefully placed in plastic buckets and slight pressed with wooden top made according to the size of the bucket. The surface moisture was removed with gentle air blower. The fruits were then shifted to cold storage at 5±1oC with 60-70% relative humidity for a period of 150 days. The calcium content of fruit was recorded as described in experiment No. 2. The experiment was laid out in completely randomized design (CRD) with thirty two treatment combinations repeated three times. The detail of the post-harvest experiment is as under Storage duration (S) = (0 and 150 days)

CaCl2 concentration = (0, 3, 6 and 9%) Dipping duration = (3, 6, 9 and 12 minutes) The data were recorded and statistically analyzed for the following post harvest quality parameters as described in experiment No. 1 at 0 and 150 days of storage. Weight loss (%), Percent juice content, Starch content, Total soluble solids (0Brix), Total sugars (%), Percent acidity, TSS/Acid ratio, Ascorbic acid (mg/100g), Fruit flesh firmness (kg/cm2), Density of fruit (g/cm3), Bitter pit (%) and Soft rot (%)

Statistical Procedures The data calculated on different parameters were subjected to Analysis of Variance (ANOVA) technique to observe the differences between the different treatment as well as their interactions. In cases where the differences were significant, the means were further assessed for differences through Least Significant Difference (LSD) test. Statistical computer software, MSTATC (Michigan State University, USA), was applied for computing both the ANOVA and LSD.

5.3. RESULTS Percent weight loss The data regarding percent weight loss revealed that there were significant variations among storage duration, CaCl2 concentration of dipping solution and dipping duration of CaCl2 solution. The interaction of storage × CaCl2 concentration, storage × dipping duration, and CaCl2 concentration × dipping duration was also significant (Table 5.1). Storage of apple fruits for 150 days resulted in significant weight loss of apple fruits. The maximum weight loss (3.24%) observed after 150 days storage. The weight loss significantly decreased with increase in CaCl2 concentration. The maximum weight loss (1.95%) observed in apple fruit at 0% CaCl2 concentration, followed by 3 and 6% with 1.68 and 1.55% respectively. The least weight loss (1.31%) recorded with 9%

CaCl2 concentration. Dipping durations also significantly affected the weight loss. The weight loss decreased with increase in dipping duration. The maximum weight loss (1.81%) recorded with dipping duration of 3 minutes, which decreased to 1.69 and 1.58% when dipping duration was extended to 6 and 9 minutes respectively and finally declined to 1.41% with dipping duration of 12 minutes. The interaction effect of storage and CaCl2 concentration was also significant. The maximum weight loss (3.91%) in apple fruits observed at 0% CaCl2 concentration after 150 days storage (Figure 5.1) as compared to 2.61% recorded with

9% CaCl2 concentration. The interaction of storage and dipping duration significantly affected the percent weight loss. The maximum weight loss (3.62%) recorded at dipping duration of 3 minutes for 150 days stored apple fruits (Figure 5.2) in contrast to 2.82% when dipping duration was extended to 12 minutes. Percent weight loss of apple fruits also significantly affected by the interaction of CaCl2 concentration and dipping duration. The maximum weight loss (2.03%) observed in fruits treated with

0% CaCl2 concentration for 3 minutes, while the minimum weight loss (0.90%) observed with 9% CaCl2 concentration for 12 minutes (Figure 5.3).

Juice content (%)

The data revealed that there was a significant effect of storage, CaCl2 concentration of dipping solution and interaction of storage × CaCl2 concentration on percent juice of apple fruit (Table 5.1). The juice content significantly decreased from 69.72% observed at 0 day storage to 55.37% recorded for fruit after 150 days storage. The juice content was significantly

high with high CaCl2 concentration. The maximum juice content (64.34%) recorded with 9% CaCl2 concentration, followed by 6 and 3% CaCl2 concentrations with juice content of 63.21 and 62.00% respectively, however, the effect was non significant between later two concentrations. The minimum juice content (60.63%) observed in fruits treated with 0% CaCl2 concentration. The interaction effect of storage and

CaCl2 concentration was also significant. The maximum juice content (70.10%) in apple fruits observed at 9% CaCl2 concentration at 0 days storage which declined to

51.84 with 0% CaCl2 concentration after 150 days storage (Figure 5.4).

Loss of starch content (Score)

Starch score of apple fruit significantly affected by storage, CaCl2 concentration of dipping solution and dipping duration of CaCl2 solution. The interaction of storage ×

CaCl2 concentration, storage × dipping duration, and CaCl2 concentration × dipping duration was also significant (Table 5.1). The starch content score decreased significantly during storage from 7.09 for fresh harvested fruit to 3.70 for fruit stored for 150 days. Starch score of apple fruit significantly increased with incremental increased in CaCl2 concentration. The maximum starch score (5.68) recorded for fruits treated with 9% CaCl2 solution, followed by 6 and 3% CaCl2 solution with 5.54 and 5.30 scores respectively. The lowest starch score (5.05) observed for apple fruits dipped in control (0% CaCl2).

Dipping durations of CaCl2 solution also significantly affected the starch content with the maximum starch content score (5.50) recorded for fruit dipped for 12 minutes in

CaCl2 solution, followed by 9 minutes with 5.44, however these two durations were at par with each other. The minimum starch content score (5.30) recorded for fruit dipped for 3 minutes in CaCl2 solution, followed by 6 minutes with 5.35 score, however the difference in these two durations were non significant (Table 5.1). The interaction effect of storage and CaCl2 concentration was also significant with the highest starch score (7.25) observed for 9% CaCl2 concentration at 0 day storage, while the lowest starch score (3.20) recorded for 0% CaCl2 concentration after 150 days storage (Figure 5.5). The interaction effect of storage and dipping duration was also significant. The maximum starch score (7.12) observed for 12 minutes dipping duration at 0 day storage, while the minimum starch score (3.54) recorded for dipping duration of 3 minutes after 150 days storage (Figure 5.6). Starch score of apple fruits also significantly affected by the interaction of CaCl2 concentration and dipping

duration. The maximum starch score (5.06) observed in fruits treated with 0% CaCl2 concentration for 6 and 9 minutes, while the minimum starch score (5.86) observed with 9% CaCl2 concentration for 12 minutes (Figure 5.7).

Total soluble solids The data revealed that total soluble solids were significantly affected by storage,

CaCl2 concentration of dipping solution and interaction of storage × CaCl2 concentration and storage × dipping duration (Table 5.1). The total soluble solids increased significantly from 10.61% recorded with 0 day storage to 13.37% recorded for fruit after 150 days storage. The total soluble solids decreased significantly with increase in CaCl2 concentration. The maximum total soluble solids (12.08%) recorded with 3 % CaCl2 concentration, followed by 0 and

6% CaCl2 concentrations with total soluble solids of 12.01 and 12.00%, with the difference being non significant between later 0 and 6% CaCal2 concentrations. The minimum total soluble solids (11.88%) recorded with 9% CaCl2 concentration. The interaction effect of storage and CaCl2 concentration was also significant. The maximum total soluble solids (13.54%) observed for 0 and 3% CaCl2 concentration at 150 days storage, while the minimum total soluble solids (10.48%) recorded for 0%

CaCl2 concentration for fresh harvested fruits (Figure 5.8). The interaction effect of storage and dipping duration was also significant. The maximum total soluble solids (13.54%) observed for 3 minutes dipping duration after 150 days storage, while the minimum total soluble solids (10.57) recorded for dipping duration of 3 minutes at 0 day storage (Figure 5.9).

Total sugar (%)

It is clear from Table 5.1 that total sugar significantly affected by storage, CaCl2 concentration of dipping solution and interaction of storage × CaCl2 concentration.

However, dipping duration in CaCl2 solution and all other interaction effect was non significant. The total sugar increased significantly during storage from 9.77% for fresh harvested fruit to 12.05% for fruit stored for 150 days. Total sugar of apple fruit significantly decreased with increase in CaCl2 concentration. The maximum total sugar (11.00%) recorded for fruits treated with 3% CaCl2 solution, followed by 0 and 6% CaCl2 solution with 10.95 and 10.93% respectively, however the differences among these

three durations was non significant. The lowest total sugar (10.76%) observed for apple fruits dipped in 9% CaCl2. The interaction effect of storage and CaCl2 concentration was also significant. The maximum total sugar (12.24%) observed for

3% CaCl2 concentration at 150 days storage, while the minimum total sugar (9.71%) recorded for 0% CaCl2 concentration for fresh harvested fruits (Figure 5.10).

Titratable acidity (%) The percent titratable acidity significantly affected by storage for 150 days and interaction of storage × CaCl2 concentration and storage × dipping duration (Table 5.1). The titratable acidity decreased significantly during storage from 0.57% for fresh harvested fruit to 0.40% for fruit stored for 150 days. The interaction effect of storage and CaCl2 concentration was significant with the highest titratable acidity (0.60%) observed for 0% CaCl2 concentration at 0 day storage, while the lowest titratable acidity (0.35%) recorded for 0% CaCl2, (Figure 5.11). The interaction effect of storage and dipping duration was also significant. The maximum titratable acidity (0.59%) observed for 3 minutes dipping duration at 0 day storage, while the minimum titratable acidity (0.38) recorded for dipping duration of 3 minutes after 150 days storage (Figure 5.12).

TSS/Acid ratio

The data on TSS/Acid ratio significantly affected by storage duration, CaCl2 concentrations of dipping solution and dipping durations in CaCl2 solution. The interaction effect of storage × CaCl2 concentration, storage × dipping duration and storage × CaCl2 concentration × dipping duration was also significant (Table 5.1). The TSS/Acid ratio increased significantly during storage from 18.75 for fresh harvested fruit to 33.82 after 150 days storage. TSS/Acid ratio of apple fruit significantly decreased with increase in CaCl2 concentration. The maximum

TSS/Acid ratio (28.09) recorded for fruits treated with 0% CaCl2 solution, followed by 3% CaCl2 solution with 27.66, however the differences between these two concentration was non significant. The lowest TSS/Acid ratio (23.88) observed for apple fruits dipped in 9% CaCl2, followed by 6% CaCl2 with 25.52, however these two concentrations was at par with each other. Dipping durations of CaCl2 solution also significantly affected the TSS/Acid ratio with the maximum TSS/Acid ratio

(27.21) recorded for fruit dipped for 3 minutes in CaCl2 solution, followed by 6 and 9 minutes with 26.73 and 25.95 respectively, however these three durations were non significant. The minimum TSS/Acid ratio (25.27) recorded for fruit dipped for 12 minutes in CaCl2 solution. The interaction effect of storage and CaCl2 concentration was also significant. The maximum TSS/Acid ratio (38.48) in apple fruits observed at

0% CaCl2 concentration after 150 days storage, whereas the minimum TSS/Acid ratio

(17.71) recorded for 0% CaCl2 concentration in fresh harvested fruits (Figure 5.13). The interaction effect of storage and dipping duration was also significant with the maximum TSS/Acid ratio (36.34) observed for 3 minutes dipping duration after 150 days storage while the minimum TSS/Acid ratio (18.07) recorded for dipping duration of 3 minutes at 0 day storage (Figure 5.14). The interaction of storage, CaCl2 concentration and dipping duration was also significant with the maximum TSS/Acid ratio (40.80) observed in fruits treated with 3% CaCl2 concentration for 3 minutes at 150 days storage, while the minimum TSS/Acid ratio (17.61) recorded in fruits treated with 0% CaCl2 concentration for 3 minutes at 0 day storage (Figure 5.15).

Table 5.1. The influence of storage, dipping duration and CaCl2 concentration on weight lost (%), percent juice, starch, TSS and total sugar (%), percent acidity and TSS/Acid ratio of apple cultivars

Storage Weight Juice Starch TSS Total Perce TSS/Aci duration loss content (Score) (0Brix) sugar nt d ratio (days) (%) (%) (%) acidit y 0 0.00 69.72 7.09 10.61 9.77 0.57 18.75 150 3.24 55.37 3.70 13.37 12.05 0.40 33.82 Significance * * * * * * * level CaCl2 (%) 0 1.95 a 60.63 b 5.05 d 12.01 ab 10.95 ab 0.48 28.09 a 3 1.68 b 62.00 ab 5.30 c 12.08 a 11.00 a 0.47 27.66 a 6 1.55 c 63.21 ab 5.54 b 12.00 ab 10.93 ab 0.49 25.52 b 9 1.31 d 64.34 a 5.68 a 11.88 b 10.76 b 0.51 23.88 b LSD at α 0.05 0.12 2.59 0.08 0.16 0.22 ns 1.87 Dipping duration (min.) 3 1.81 a 61.91 5.30 b 12.06 10.98 0.48 27.21 a 6 1.69 b 62.50 5.35 b 12.02 10.97 0.48 26.73 ab 9 1.58 b 62.74 5.44 a 11.96 10.90 0.49 25.95 ab 12 1.41 c 63.03 5.50 a 11.94 10.80 0.50 25.27 b LSD at α 0.05 0.12 ns 0.08 ns ns ns 1.87 Interactions Significance Level S × Ca * * * * * * * S × D * ns * * ns * * Ca × D * ns * ns ns ns ns S × Ca × D ns ns ns ns ns ns *

Mean followed by similar letter(s) in column do not differ significantly ns = Non Significant * = Significant at 5 % level of probability. S × Ca = Interaction of storage duration and CaCl2 concentration S × D = Interaction of storage and dipping duration, Ca × D = Interaction of CaCl2 concentration and dipping duration S × Ca × D = Interaction of storage duration, CaCl2 concentration and dipping duration

4.5 4 3.5 3 2.5 2

1.5 Weight Loss (%) Loss Weight 1 0.5 0 0 3 6 9

CaCl2 (%)

Figure 5.1 Influence of CaCl2 concentrations on percent weight loss in apple fruits stored for 150 days

4 3.5 3 2.5 2

1.5 Weight Loss (%) Loss Weight 1 0.5 0 3 6 9 12 Dipping Durations (Minutes)

Figure 5.2 Influence of dipping durations on percent weight loss in apple fruits stored for 150 days

2.3 2.1 1.9 1.7 1.5 3 6 1.3 9

Weight loss(%) Weight 1.1 12 0.9 0.7 0.5 0 3 6 9

CaCl2 (%)

Figure 5.3 Interaction effect of CaCl2 concentration and dipping duration on percent weight loss in apple fruit

80 70 60 50 0 40 150

30 Juice Content(%) Juice 20 10 0 0 3 6 9

CaCl2 (%)

Figure 5.4 Interaction effect of CaCl2 concentrations and storage on percent juice of apple fruit

8 7 6 5 0 4 150

Starch (Scores) Starch 3 2 1 0 0 3 6 9

CaCl2 (%)

Figure 5.5 Interaction effect of CaCl2 concentration and storage on starch score of apple

8

7

6

5 0

4 150

Starch (Scores) Starch 3

2

1

0 3 6 9 12 Dipping Durations (Minutes)

Figure 5.6 Interaction effect of dipping duration and storage on starch scores of apple

6.2

6

5.8

3 5.6 6 5.4

9 Starch (scores) Starch 5.2 12

5

4.8 0 3 6 9

CaCl2 (%)

Figure 5.7 Interaction effect of CaCl2 concentration and dipping duration on starch scores of apple

16

14

12

10 0 8 150 6

4 Total SolubleSolids Total 2

0 0 3 6 9

CaCl2 (%)

Figure 5.8 Interaction effect of CaCl2 concentration and storage duration on TSS of apple

16

14

12

10 0

8 150

6

4 Total Soluble Solids (%) SolubleSolids Total 2

0 3 6 9 12 Dipping Durations (Minutes)

Figure 5.9 Interaction effect of dipping duration and storage duration on TSS of apple

14 12 10 8 0 6 150

Total Sugars (%) Sugars Total 4 2 0 0 3 6 9

CaCl2 (%)

Figure 5.10 Interaction effect of CaCl2 concentration and storage duration on total sugar (%) of apple

0.7

0.6

0.5

0.4 0

0.3 150

0.2 Titratable Acidity (%) Acidity Titratable 0.1

0 0 3 6 9

CaCl2 (%)

Figure 5.11 Interaction effect of CaCl2 concentration and storage duration on titratable acidity (%) of apple

0.7

0.6

0.5

0.4 0

0.3 150

0.2 Titratable Acidity (%) Acidity Titratable 0.1

0 3 6 9 12 Dipping Durations (Minutes)

Figure 5.12 Interaction effect of dipping duration and storage duration on titratable acidity (%) of apple

45 40 35 30 25 0 20 150

15 TSS/Acid Ratio TSS/Acid 10 5 0 0 3 6 9 CaCl2 (%)

Figure 5.13 Interaction effect of CaCl2 concentration and storage duration on TSS/Acid ratio of apple

45 40 35 30 0 25 20 150

15 TSS/Acid Ratio TSS/Acid 10 5 0 3 6 9 12 Dipping Durations (Minutes)

Figure 5.14 Interaction effect of dipping duration and storage duration on TSS/Acid ratio of apple

50

45

40

35 3 30 6

TSS/Acid Ratio TSS/Acid 25 9 20 12 15

10

5

0 0 3 6 9 0 3 6 9

0 0 0 0 150 150 150 150

CaCl2 (%) and Storage Durations (Days)

Figure 5.15 Interaction effect of CaCl2 concentrations, dipping durations and storage durations on TSS/Acid ratio of apple

Ascorbic acid (mg/100g)

The data in table 3.2 reveal that ascorbic acid significantly affected by storage, CaCl2 concentration of dipping solution and interaction of storage × CaCl2 concentration (Table 5.2). The ascorbic acid content of apple fruit decreased significantly from 15.14 mg/100g observed at 0 day storage to 11.58 mg/100g recorded in fruits stored for 150 days.

The ascorbic acid was significantly high with higher CaCl2 concentration. The maximum ascorbic acid (13.87 mg/100g) recorded with 9% CaCl2 concentration, followed by 6 and 3% CaCl2 concentrations with ascorbic acid of 13.61 and 13.29 mg/100g respectively. The difference in 3 and 6% was, however, non significant. The lowest ascorbic acid (12.67 mg/100g) observed in fruits treated with 0% CaCl2 concentration. The interaction effect of storage and CaCl2 concentration was also significant. The maximum ascorbic acid (15.18 mg/100g) observed for 9% CaCl2 concentration at 0 days storage, while the minimum ascorbic acid (10.28) recorded for

0% CaCl2 concentration at 150 days storage (Figure 5.16).

Fruit flesh firmness (kg/cm2)

The effect of storage duration, CaCl2 concentration of dipping solution and dipping duration in CaCl2 solution was significant on fruit flesh firmness of apple fruits. The interaction of storage × CaCl2 concentration and storage × dipping duration was also significant (Table 5.2). The fruit flesh firmness of apple fruit decreased significantly by storage. It decreased from 7.30 kg/cm2 for fresh harvested fruits to 5.27 kg/cm2 after 150 days storage. The fruit flesh firmness of the fruit remained significantly higher with increasing CaCl2 concentration. The maximum fruit flesh firmness (6.54 kg/cm2) observed in apple 2 fruit at 9% CaCl2 concentration, followed by 6 and 3% with 6.41 and 6.22 kg/cm 2 respectively. The least fruit flesh firmness (5.98 kg/cm ) recorded with 0% CaCl2 concentration. Dipping durations also had significant effect on the fruit flesh firmness. The fruit flesh firmness was high with longer dipping durations. The lowest fruit flesh firmness (6.13 kg/cm2) observed at dipping duration of 3 minutes, which increased significantly to 6.18 and 6.35 kg/cm2 with 6 and 9 minutes dipping duration and finally to the maximum fruit flesh firmness (6.49 kg/cm2) recorded with dipping duration of 12 minutes (Table 5.2). The interaction effect of storage and CaCl2 concentration was also significant. The maximum fruit flesh firmness (7.44 kg/cm2)

observed for 9% CaCl2 concentration at 0 days storage, while the minimum ascorbic 2 acid (4.81 kg/cm ) recorded for 0% CaCl2 concentration at 150 days storage (Figure 5.17). The interaction effect of storage and dipping duration was also significant with the maximum fruit flesh firmness (7.36 kg/cm2) observed for 12 minutes dipping duration at 0 day storage, while the minimum fruit flesh firmness (5.03 kg/cm2) recorded for dipping duration of 3 minutes after 150 days storage (Figure 5.18).

Fruit density (g/cm3)

It is clear from table 5.2 that fruit density significantly affected by storage, CaCl2 concentration of dipping solution and dipping duration in CaCl2 solution. The interaction of storage × CaCl2 concentration was also significant (Table 5.2). The fruit density decreased significantly during storage from 0.85 g/cm3 for fresh harvested fruit to 0.80 g/cm3 for fruit stored for 150 days. Fruit density of apple was significantly high with increase in CaCl2 concentration. The highest fruit density (0.84 3 g/cm ) recorded for fruits treated with 9% CaCl2 solution, followed by 6% CaCl2 solution with 0.83 g/cm3, however the differences between these two concentration was non significant. The lowest fruit density (0.81 g/cm3) observed for apple fruits dipped in 0% CaCl2 concentration, followed by 3% CaCl2 concentration with 0.82 g/cm3, however the effect was non significant. The fruit density of apple fruit significantly increased with increase in dipping duration. The minimum fruit density 3 (0.82 g/cm ) recorded for fruits dipped for 3 minutes in CaCl2 solution increased to 3 the maximum (0.84 g/cm ) in fruit dipped for 12 minutes in CaCl2 solution while it was 0.83 and 0.82 g/cm3 for 6 and 4 minutes respectively (Table 5.2). The interaction effect of storage and CaCl2 concentration was also significant. The maximum fruit 3 density (0.85 g/cm ) observed for 0 to 9% CaCl2 concentration at 0 days storage, 3 while the minimum fruit density (0.78 g/cm ) recorded for 0% CaCl2 concentration at 150 days storage (Figure 5.19).

Bitter pit (%)

The effect of storage duration, CaCl2 concentration of dipping solution and dipping duration in CaCl2 solution significantly affected the bitter pit incidence of apple fruits.

The interaction of storage × CaCl2 concentration, storage × dipping duration, CaCl2 concentration × dipping duration and storage × CaCl2 concentration × dipping duration was also significant (Table 5.2). The incidence of bitter pit on apple fruit increased significantly to 16.95% after 150 days storage. The influence of CaCl2 concentration revealed that the incidence of bitter pit decreased significantly with increase in CaCl2 concentration. The highest bitter pit incidence (15.18%) observed on apple fruit at 0% CaCl2 concentration, which decreased to 6.06% and 3.80% with CaCl2 concentration of 6 and 9%. The incidence of bitter pit was significantly affected by dipping durations. The bitter pit incidence decreased with increase in dipping duration. The maximum bitter pit (11.92%) recorded with dipping duration of 3 minutes decreased to 9.31 and 7.08% with 6 and 9 minutes dipping duration respectively. Extending the dipping duration to 12 minutes decreased the bitter pit incidence to the minimum of 5.60% (Table 5.2).

The interaction effect of storage and CaCl2 concentration was also significant. The maximum incidence of bitter pit (30.36%) in apple fruits observed at 0% CaCl2 concentration after 150 days storage (Figure 5.20). The interaction effect of storage and dipping duration was also significant with the maximum bitter pit incidence (23.84%) observed for 3 minutes dipping duration while the minimum bitter pit (11.19%) recorded for dipping duration of 12 minutes after 150 days storage (Figure

5.21). Incidence of bitter pit was also significantly affected by the interaction of CaCl2 concentration and dipping duration. The maximum bitter pit incidence (15.76%), observed in fruits treated with 0% CaCl2 concentration for 6 minutes, while the minimum weight loss (1.25%) observed with 9% CaCl2 concentration for 12 minutes

(Figure 5.22). The interaction of storage, CaCl2 concentration and dipping duration was also significant with the maximum bitter pit incidence (31.52%) observed in fruits treated with 0% CaCl2 concentration for 3 minutes after 150 days storage (Figure 5.23).

Soft rot (%)

The effect of storage duration, CaCl2 concentration of dipping solution and duration of dipping in CaCl2 solution also significantly affected the soft rot incidence of apple

fruits. The interaction of storage × CaCl2 concentration and storage × dipping duration was also significant (Table 5.2). The incidence of soft rot in apple fruit increased significantly to 13.44% after 150 days storage. The soft rot significantly decreased with incremental increase in CaCl2 concentration. The highest soft rot incidence (15.33%) observed in apple fruit at 0%

CaCl2 concentration, decreased to 6.04 and 3.43% with 3 and 6% solution respectively and finally reached to the minimum of 2.10% with 9% CaCl2 concentration. Increasing dipping durations also decreased the incidence of soft rot significantly from the maximum of 7.07% with dipping duration of 3 minutes to 6.94 and 6.60% with 6 and 9 minutes dipping respectively. The lowest soft rot incidence (6.28%) observed at dipping duration of 12 minutes. The interaction effect of storage and CaCl2 concentration was also significant with the highest soft rot incidence

(30.65%) recorded on apple fruits with 0% CaCl2 concentration after 150 days storage (Figure 5.24). The interaction effect of storage and dipping duration was significant. Soft rot incidence was the maximum (14.14%) on apple fruits dipped for 3 minutes after 150 days storage (Figure 5.25) as compared to the minimum of 12.55% observed with 12 minutes dipping duration for the same storage duration.

Table 5.2. The influence of storage, dipping duration and CaCl2 concentration on ascorbic acid (mg/100g), fruit flesh firmness (kg/cm2), fruit density (g/cm3), bitter pit (%) and soft rot (%) of apple cultivars

Storage duration (days) Ascorbic acid Fruit flesh Fruit density Bitter pit (%) Soft rot (%) (mg/100g) firmness (g/cm3) (kg/cm2) 0 15.14 7.30 0.85 0.00 0.00

150 11.58 5.27 0.80 16.95 13.44 Significance level * * * * *

CaCl2 (%) 0 12.67 b 5.98 c 0.81 b 15.18 a 15.33 a 3 13.29 ab 6.22 b 0.82 b 8.87 b 6.04 b

6 13.61 a 6.41 ab 0.83 a 6.06 c 3.43 c

9 13.87 a 6.54 a 0.84 a 3.80 c 2.10 d LSD at α 0.05 0.68 0.20 0.01 2.53 0.45 Dipping duration (minutes) 3 13.15 6.13 c 0.82 c 11.92 a 7.07 a 6 13.27 6.18 bc 0.82 bc 9.31 b 6.94 ab

9 13.42 6.35 ab 0.83 ab 7.08 bc 6.60 bc 12 13.60 6.49 a 0.84 a 5.60 c 6.28 c LSD at α 0.05 ns 0.20 0.01 2.53 0.45 Interactions Significance Level S × Ca * * * * * S × D ns * ns * * Ca × D ns ns ns * ns S × Ca × D ns ns ns * ns

Mean followed by similar letter(s) in column do not differ significantly ns = Non Significant * = Significant at 5 % level of probability. S × Ca = Interaction of storage duration and CaCl2 concentration S × D = Interaction of storage and dipping duration Ca × D = Interaction of CaCl2 concentration and dipping duration S × Ca × D = Interaction of storage duration, CaCl2 concentration and dipping duration

18 16 14 12 10 0 8 150 6

Ascorbic acid (mg/100g) acid Ascorbic 4 2 0 0 3 6 9 CaCl2 (%)

Figure 5.16 Interaction effect of CaCl2 concentration and storage duration on ascorbic acid (mg/100g) of apple

9

8 ) 2 7 6 0 5 150 4

Firmness (kg/cm Firmness 3 2 1 0 0 3 6 9 CaCl2 (%)

Figure 5.17 Interaction effect of CaCl2 concentration and storage duration on fruit flesh firmness (kg/cm2) of apple

9 8

7 ) 2 6 0 5 15 4 0 3

Firmness (kg/cm Firmness 2 1 0 3 6 9 12 Dipping Durations (Minutes)

Figure 5.18 Interaction effect of dipping duration and storage duration on fruit flesh firmness (kg/cm2) of apple

0.88

0.86 ) 3 0.84 0 0.82 150 0.8

0.78

Fruit Density Density (g/cm Fruit 0.76

0.74 0 3 6 9 CaCl2 (%)

Figure 5.19 Interaction effect of CaCl2 concentration and storage duration on fruit density (g/cm3) of apple

40 35 30 25 20

15 Bitter Pit (%) Pit Bitter 10 5 0 0 3 6 9 CaCl2 (%)

Figure 5.20 Influence of CaCl2 concentrations on bitter pit (%) of apple fruits after 150 days storage

30

25

20

15

Bitter Pit (%) Pit Bitter 10

5

0 3 6 9 12 Dipping Durations (Minutes)

Figure 5.21 Influence of dipping durations on bitter pit (%) of apple fruits stored for 150 days

25

20 3 15 6 10 9

5 12 Bitter Pit (%) Pit Bitter 0 -5 0 3 6 9

CaCl2 (%)

Figure 5.22 Interaction effect of CaCl2 concentration and dipping duration on bitter pit (%) of apple

40

30 3 6 20 9

10 12 Bitter Pit (%) Pit Bitter 0 0 3 6 9 -10

CaCl2 (%)

Figure 5.23 Influence of CaCl2 concentrations and dipping durations on bitter pit (%) of apple fruits stored for 150 days

35

30

25

20

15 Soft Rot (%) Rot Soft 10

5

0 0 3 6 9

CaCl2 (%)

Figure 5.24 Effect of CaCl2 concentrations on soft rot (%) incidence in apple fruits stored for 150 days

15

14.5

14

13.5

13

Soft Rot (%) Rot Soft 12.5

12

11.5

11 3 6 9 12 Dipping Durations (Minutes)

Figure 5.25 Influence of dipping durations on soft rot (%) incidence in apple fruits after 150 days storage

Calcium content of the fruit The data in table 5.3 revealed that apple cultivar Red Delicious varied significantly in calcium content of the fruit. Incremental increase was observed in calcium content of apple fruit, with gradual increased in calcium chloride concentration as well as dipping duration. The calcium content of fruit was increased from 36 mg /kg observed in control to the maximum of 88 mg/kg recorded in fruits treated dipped in 9% CaCl2 solution for 12 minutes.

Table 5.3. Calcium content (mg/kg) of apple fruit cv. Red Delicious as affect

by CaCl2 concentration and dipping duration

CaCl2 concentration (%) Dipping duration 0 3 6 9 3 36 45 54 59 6 36 51 62 68 9 36 54 67 76 12 36 65 74 88

5.4. DISCUSSION

Percent weight loss and Juice content (%) The moisture content of fruits is important quality criteria (Gorini et al., 1979) and the loss of turgor pressure and subsequent softening is depends on moisture loss (Vander- Beng, 1981). The data regarding percent weight loss and juice content revealed that storage duration, CaCl2 concentration of dipping solution and dipping duration of

CaCl2 solution resulted in significant variations in weight loss of apple fruit. Storage of apple for 150 days resulted in significant weight loss of apple fruits. The weight loss depends on water present in the fruit and the structure of the skin and nature of waxes on the surface (Babos et al., 1984; Veravrbeke et al., 2003). The weight loss in fruits increased linearly with increase in storage duration due water loss and respiration (Blampired, 1981; El-Shennawi, 1989; Gavlheiro et al., 2003; Erturk, 2003; Ghafir et al., 2009). The weight loss decreased significantly with increase in

CaCl2 concentration or dipping solution in CaCl2 solution (Table 5.1).

The decrease in weight loss with increase in CaCl2 concentration or dipping duration is in accordance with Ashore, (2000) and Hayat et al. (2003). The juice content generally declined with increasing storage duration (Allan et al., 2003) (Table 5.1). The juice content significantly decreased from by 20.58% after 150 days storage. The juice content of apple fruit depends mainly on the water content of the fruit and decreasing the rate of water loss increases the juice content (Dzonova et al., 1970).

The juice content was significantly high with high CaCl2 concentration of 9% (3.78%) as compared to control. While dipping duration for 12 minutes also increased the juice content by 1.78%, it was non significant with other durations (Table 5.1)

Loss of starch content (Score) The starch is the major storage carbohydrates in apple fruit (Beaudry et al., 1989). Starch score of apple fruit decreased significantly by 47.81% with storage for 150 days, but CaCl2 concentrations of dipping solution 9% and dipping duration in calcium solution and dipping duration for 12 minutes resulted in 12.8 and 3.64% higher starch score (Table 5.1). The starch content of apple fruit decreased significantly during storage. Since starch is the major storage carbohydrates in apple fruit (Beaudry et al., 1989), it is converted to sugars at the onset of ripening and during storage to meet the respiratory demand of the fruit (Bidabe et al., 1970;

Crouch, 2003; Saleh et al., 2009), thus it is likely to observe the decline in starch content during storage of apple fruit. The apple fruit store its extra sugars in the form of starch (Beaudry et al., 1989) which is converted to free sugars to meet the respiratory demands. Since the application of calcium delay the overall ripening of apple fruit, the loss of starch content is also less with CaCl2 application (Kadir, 2005).

Total soluble solids (%) The total soluble solids content is an important quality parameter (Daillant et al., 1996) and relates to soluble sugars and other solutes in the juice (Echeveria et al., 2002). Total soluble solids of apple for 150 days resulted in 20.64% increase in total soluble solids content and its increase was retarded by different CaCl2 concentrations of dipping solution. The total soluble solids decreased significantly with increase in

CaCl2 concentration, it decreased by 1.08% with 9% CaCl2 concentration (Table 5.1). The increase in TSS may be due to the enzymatic conversion of higher polysaccharides such as starches to simple sugars during ripening (Hussain et al.,

2008). The less increase in TSS with increasing CaCl2 concentrations may be due to the delay in natural physiological processes like ripening and senescence by CaCl2, due to the inhibition or retardation of conversion to simple sugars from complex polysacharides (Izumi and Watada 1994; Agar et al., 1999; Rosen and Kader, 1989; Kader, 1986). It is however, also important that the loss of moisture may concentrate the cell sap, resulting in low juice content but high TSS percentage. The total soluble solids generally increase during storage (Mahajan, 1994; Rivera, 2005) and calcium treatment increases the calcium content in the fruit and decreases the storage related loss of texture and other quality components including TSS (Mahajan, 1994; Benavides et al., 2002; Rivera, 2005). Though acidity increased but not significantly while the TSS decreased significantly with calcium application, thus, resulted in relatively lower TSS/Acid ratio.

Total sugar (%) The total sugars content of apple fruit increased by 18.92% with storage for 150 days.

The application of CaCl2 concentration of dipping solution and dipping duration in

CaCl2 solution decreased the total sugars content of apple fruit. Increasing CaCl2 concentration resulted in 1.73% decline in total sugars with 9% CaCl2 concentration (Table 5.1). The sugars content of apple fruit contribute to the fruit sweetness, and,

thus, is a major fruit quality characteristics. In apple fruit the sugars tend to increase with maturation (Wani et al., 2008). The apple fruit accumulate starch at the early stages of maturation, which is hydrolyzed to sugars at edible maturity (Magein and Leurquin, 2000) and during storage (Beaudry et al., 1989), resulting in increased total sugars with increased storage duration (Bidabe et al., 1970; Crouch, 2003). Since the application of Ca delays the changes associated with ripening and senescence in apple fruits such as increased in total sugars (Wills et al., 1977; Goncalves et al., 2000), its application resulted in lower total sugars with increasing CaCl2 concentration probably by retarding the general senescence or decreasing the water loss (Hayat et al., 2003).

Titratable acidity (%) The acidity of the apple fruit is due to different organic acid present in the fruit, with malic acid being predominant (Bilisili et al., 1970). The titratable acidity of apple fruit decreased significantly by 29.82% with storage for 150 days but was not significantly influenced by CaCl2 concentration as well as dipping duration (Table 5.1). The decline in titratable acidity depends on the rate of metabolism (Murata and Minamide, 1970; Clarke et al., 2001) especially respiration which consumed organic acid and thus decline acidity (Rivera, 2005; Ghafir et al., 2009). The decrease in acidity also results due to the utilization of organic acids and raw materials for synthesis of other compounds during ripening, because the organic acids are consumed in respiration, resulting lower acidity with increasing storage duration (Rivera, 2005; Ghafir et al.,

2009).

TSS/Acid ratio The TSS/Acid ratio was increased by 44.56% with storage duration for 150 days. The total soluble solids decreased significantly with increase in CaCl2 concentration and dipping duration it decreased by 14.98 and 7.12% with 9% CaCl2 concentration and

12 minutes dipping duration in CaCl2 solution (Table 5.1). The TSS/Acid ratio of apple and other fruits is a major quality parameter (Weibel et al., 2004; Peck et al., 2006). The TSS/Acid ratio generally increases with increasing storage duration. The increase in TSS/Acid ratio can be attributed to starch breakdown resulting in free sugars (Beaudry et al., 1989) and decline in organic acids due to its consumption in respiration (Mahajan, 1994; Rivera, 2005; Ghafir et al., 2009). Since the increase in

TSS was retarded by the calcium treatments (Mahajan, 1994; Benavides et al., 2002; Rivera, 2005) while the acidity was retained at high levels, it resulted in lower

TSS/Acid ratio with increasing CaCl2 concentration or dipping duration.

Ascorbic acid (mg/100g) The ascorbic acid content of the fruit decreased significantly with storage (23.51%).

The application of different CaCl2 concentration significantly affected the ascorbic acid content of the fruit but dipping duration in CaCl2 solution had no significant effect. The fruit treated with 9% CaCl2 solution had 8.65% higher ascorbic acid (Table 5.2). Ascorbic acid is usually considered as an index of nutrient quality in apple fruit. Ascorbic acid is a bioactive compound having antioxidant properties (Lata, 2007). The ascorbic acid decreased significantly with storage (Purvis, 1983; Hayat et al., 2003). The ascorbic acid is a high labile vitamin which tends to decline during storage (Adisa, 1986). The ascorbic acid loss during storage is known to be due to its antioxidant activity especially under postharvest storage conditions (Davey et al., 2000). The ascorbic acid can be irreversible oxidized (Parviainen and Nyyssonen, 1992; Pardio-Sedas et al., 1994), thus causing a decrease during storage (Jung and Watkins, 2008). The retention of relatively high ascorbic acid with increasing CaCl2 concentrations or duration may due to the regulation of oxidative processes is the cytosol responsible for ascorbic acid degradation (Faust and Shear 1972).

Fruit flesh firmness (kg/cm2) Fruit flesh firmness is an important criterion that determines edible quality and market value of apples and other fruits (Stow, 1995; De-Ell et al., 2001, Pocharski et al., 2000, Weibel et al., 2004; Peck et al., 2006). The fruit flesh firmness of apple fruit decreased by 27.8% with storage for 150 days but was retained at the maximum with

9% CaCl2 concentration which was 8.56% more firmer than control. Dipping durations also had significant effect on the fruit flesh firmness with the maximum fruit flesh firmness retained with dipping in CaCl2 solution for 12 minutes, which was 5.5% higher than capered than dipping for 3 minutes (Table 5.2). The loss of fruit flesh firmness is a serious problems resulting in quality losses (Kov et al., 2005). The fruit flesh firmness of the apple fruit is due to texture of the flesh and textural changes of fruits especially the cell wall breakdown (Fuller, 2008) due to enzymatic activities

(Yamaki and Matsuda, 1977) and pectin solubalization (Bartley et al., 1982; Jackman and Stanley, 1995; Chang-Hai et al., 2006), which reduces the mechanical strength of cell walls and decrease the fruit flesh firmness in apple fruits (Kov et al., 2003; Kov et al., 2005). The retention of fruit flesh firmness with increasing calcium concentration or dipping duration can be attributed to the formation of calcium pectates leading to increased rigidity of the cell wall and, thus, improved turgor pressure (Jackman and Stanley, 1995; Luna-Guzman and Barrett, 2000).

Fruit density (g/cm3) The density of the fruit is a physical characteristic that has been can as maturity (Wolfe et al., 1974) and quality index. Potato where the tuber‟s density is generally correlated with the starch content of tubers (Zaltzman et al., 1987), dry matter (Wilson and Lindsay, 1969), as well as the mechanical resistance of tubers (Hudson, 1975). The density of the fruit may be influenced by rind thickness (Cohen, 1972), juice content (Zaltzman et al., 1987), dry matter (Jordan et al., 2000) total sugars and starch contents (Robert et al., 2000). Thus, fruit density also been used as maturity and quality index in many fruits and vegetables such as apricots, strawberries, tomato, pea, etc (Wolfe et al., 1974; Zaltzman et al., 1987; McGlone et al., 2007). The fruit density decreased significantly with storage of apple fruit for 150 days. There was

5.88% decline with 150 days storage. The fruit density in relation to CaCl2 concentration of dipping solution revealed that the fruit density was 3.57% higher than control (Table 5.2). Similarly the fruit density was retained higher with increasing duration in CaCl2 solution. The changes in density of apple fruit is a function air spaces and solutes dissolved in the cell sap (Marlow and Loescher, 1984; Bayindirli, 1993) Therefore, the specific gravity is high in fresh fruits and decline during storage (Sakiyama and Nakamura, 1976) due to collapse of intercellular spaces and loss of moisture (Mitropoulos and Lambrinos, 2000). Both increasing the concentration of Calcium in dipping solution or duration of dipping in calcium solution decreased the loss of specific gravity in apple fruit. The calcium regulates the fruit flesh firmness by maintaining cell wall structure (Poovaiah, 1986; Conway and Sams, 1987) and delaying the natural ripening and senescence (Rosen and Kader, 1989). Thus, the calcium may decrease the development of air spaces associated with ripening and responsible for the loss of specific gravity (Ruess and StoEsser, 1993).

Bitter pit (%) Bitter pit is a physiological disorder, characterized by depressed brown lesions in the skin of the fruit (Ferguson and Watkins, 1989). The incidence of bitter pit may be related to genetic factors but growth conditions and maturity at harvest (Crouch, 2003) or Ca concentration of the fruit (Pesis et al., 2009) and other nutrition (Fallahi et al., 1997) may regulate its development. The bitter pit incidence of apple fruit was significantly higher with 150 days storage but decreased with increasing CaCl2 concentration of dipping solution so that it was 3.99 fold higher in control as capered to 9% CaCl2 treatment. The bitter pit incidence also decreased with increasing dipping duration in CaCl2 solution so that it was 4.71 fold higher with dipping for 12 minutes as compared to 3 minutes (Table 5.2). The bitter pit incidence generally increases with increasing storage duration (Pesis et al., 2009) but can be decreased by pre (Peryea et al., 2003) and postharvest Ca application (Reid and Padfield, 1975; Jones and Higgs, 1982). Calcium helps in regulating the metabolism in apple fruit, and adequate concentration maintains fruit flesh firmness, delays fruit ripening, lower the incidence of physiological disorders such as water core, bitter pit, and internal breakdown (Bangerth, et al., 1972; Faust and Shear, 1968; Mason et al., 1975; Reid and Padfield, 1975) and suppress Erwinia carotovora (Jones) incidence on apple fruits (Conway, 1982; Sharples and Johnson, 1977). The deficiency of calcium, due to decreased movement of calcium to the fruit with maturation has been held responsible for bitter pit in apple fruit (Fuller, 1980). Thus calcium spray is an effective methods of decreasing Ca deficiency and bitter pit incidence (Dris and Niskanen, 1999, Rosenberger et al., 2001; Conway et al., 2002).

Soft rot (%) The incidence of soft rot apple fruit increased significantly with storage for 150 days but decreased with increasing CaCl2 concentration of dipping solution or increasing dipping duration in CaCl2 solution. The soft rot incidence in control fruit was 7.3 fold higher as capered to 9% CaCl2 concentration of dipping solution but it was only 1.13% higher with dipping for 12 minutes than 3 minutes (Table 5.2). Thus the incidence of different pathogens on apple fruit depends on cultivar but generally increase during storage (Kader, 1985; Spotts et al., 1999). It has been suggested that the increased susceptibility in apple fruit to physiological disorders and pathogens during storage may be due to decrease in antioxidant activities (Watkins, 2003), yet

no correlation is observed between ascorbic acid and the incidence of soft rot or bitter pit (Mattheis and Rudell, 2008). The decreased soft rot incidence with increased calcium concentration may be due to the calcium-induced delay in natural ripening and senescence (Agar et al., 1999), which causes the fruit susceptible to pathogens.

Calcium content of the fruit

Dipping of apple fruit in 9% CaCl2 solution for 12 minutes resulted in 2.44 fold increase in the calcium content of apple fruit. It indicates that dipping in calcium chloride solution is an effective way of increasing the calcium content of the fruit.

Furthermore, apple cultivars had comparable incorporation of CaCl2. Since the CaCl2 uptake is mainly through the lenticels (Dewey and Lee, 1980), it is likely to observe about similar increase in calcium content of the all the cultivars under study.

5.5. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary

The research on “influence of CaCl2 concentration and dipping duration on internal quality changes in apple cv Red Delicious” was carried out at Horticulture Postharvest Laboratory, Khyber Pakhtunkhwa Agricultural University, Pakistan during 2009-10. The fruits from apple cultivar “Red Delicious” were harvested at commercial maturity stage at Matta, Swat. The fruits were dipped in 0, 3, 6 and 9% CaCl2 solution for 3, 6, 9 and 12 minutes and shifted to cold storage for the period of 150 days. The percent weight loss, total soluble solids, total sugar, TSS/Acid ratio, bitter pit incidence and soft rot increased while, juice content, starch score, titratable acidity, ascorbic acid, fruit flesh firmness and density of fruit declined with increase in storage duration.

Different concentrations of CaCl2 significantly affected all the parameters except titratable acidity. The increase was recorded in juice content, starch score, total sugar, total soluble solids, TSS/Acid ratio, ascorbic acid, fruit flesh firmness and density of fruit with increase in CaCl2 concentration, whereas, percent weight loss, bitter pit incidence and soft rot incidence reduced with increase in CaCl2 concentration. The increase was recorded in starch score, fruit flesh firmness and density of fruit with increase in dipping duration in CaCl2 solution and percent weight loss, TSS/Acid ratio, bitter pit incidence and soft rot incidence significantly reduced with increase in dipping duration of CaCl2 solution. However, dipping duration has affected juice content, Titratable acidity, total soluble solids, total sugar and ascorbic acid non significantly.

Conclusions

o Fruits treated with 6% CaCl2 not only retained the quality attributes

significantly higher than 0 or 3% CaCl2 solution but also decreased the bitter pit incidence.

o Fruits dipped in 9% CaCl2 solution had the least weight loss and soft rot incidence. o Dipping duration of 9 minutes has not only retained the fruit quality high but also reduced the incidence of bitter pit, soft rot as well as high fruit flesh firmness and fruit density. o 12 minutes dipping duration has significantly reduced the percent weight loss. Recommendations

 Fruits treated with 6% CaCl2 not only retained the quality attributes

significantly higher than 0 or 3% CaCl2 solution but also decreased the bitter pit incidence, thus it can be recommended for retention of quality during long term storage.

 Fruits dipped in 9% CaCl2 solution have the least weight loss and soft rot incidence, therefore it is recommended to reduce the soft rot incidence and weight loss.  Dipping duration of 9 minutes is recommended for prolong storage of apple fruits because this duration has not only retained the fruit quality high but also reduced the incidence of bitter pit, soft rot as well as high fruit flesh firmness and fruit density.  12 minutes dipping duration has significantly reduced the percent weight loss, so can be recommended to reduce the weight loss during storage.

CHAPTER 6: INFLUENCE OF CaCl2 TREATMENT ON STORAGE PERFORMANCE OF APPLE CULTIVARS

Ibadullah Jan and Abdur Rab Department of Horticulture, Khyber Pakhtunkhwa Agricultural University Peshawar, Pakistan

Abstract

The research on “influence of CaCl2 treatment on storage performance of apple cultivars” was carried out at Horticulture Postharvest Laboratory, Khyber Pakhtunkhwa Agricultural University, Peshawar-Pakistan during 2009-10. The fruits were harvested from apple cultivars: Royal Gala, Mondial Gala and Golden Delicious at commercial maturity stage at Matta, Swat. The fruits were treated with 0 and 9%

CaCl2 solution for the period of 12 minutes and stored for the period of 150 days at 5±1°C with 60-70% relative humidity. Apple cultivar Royal Gala had the highest juice content (59.20%), ascorbic acid (12.88 mg/100g) and fruit flesh firmness (5.46 kg/cm2). Mondial Gala had the least weight loss (1.55%) as well as maximum titratable acidity (0.54%). The maximum bitter pit incidence (6.96%) was observed in cultivar Golden Delicious. Percent weight loss, total soluble solids, total sugar, TSS/Acid ratio, bitter pit incidence and soft rot increase while, juice content, starch score, titratable acidity, ascorbic acid, fruit flesh firmness and density of fruit declined during storage. The application CaCl2 significantly decreased percent weight loss (1.16%), total soluble solids (10.92), total sugar (10.50%), TSS/Acid ratio (21.69), bitter pit incidence (1.03%) and soft rot (1.41%), while juice content (57.58%), starch score (5.49), titratable acidity (0.53%), ascorbic acid (12.97 mg/100g), fruit flesh 2 3 firmness (5.62 kg/cm ) and density of fruit (0.78 g/cm ) increased with CaCl2 solution.

3.1. INTRODUCTION

Apple is prone to qualitative and quantitative losses after harvest, therefore it is stored in cold storage in order to minimize the losses and ensure its availability in the market thought out the year (Shah et al., 2002). The storage life is reduced by loss of fruit flesh firmness (Kov et al., 2005), loss of chemical attributes (Golias et al., 2008), physiological disorders like bitter pit (Juan et al., 1999) and disease incidence or decay (Ingle et al., 2000; Hribar et al., 1996). Thus, attempts have been made to explore different methods to reduce the postharvest losses during storage (Lau, 1992; Conway et al., 2002; Mahmud et al., 2008; Gupta and Jawandha, 2010). Calcium plays an important role in regulating the metabolism in apple fruit, and adequate concentration maintains fruit flesh firmness, delays fruit ripening, lower the incidence of physiological disorders such as water core, bitter pit, and internal breakdown (Faust and Shear, 1968; Bangerth et al., 1972; Mason et al., 1975; Reid and Padfield, 1975) and suppress Erwinia carotovora (Jones) incidence on apple fruits (Sharples and Johnson, 1977; Conway, 1982). The apple fruit grown on soil having optimum calcium level, may experienced Ca deficiency symptoms (Petersen, 1980) which may lead to several physiological disorders. The apple fruits having less than 50 mg kg−1 Ca content of fresh weight, are sensitive to physiological disorders like bitter pit and internal breakdown (Petersen, 1980). By contrast optimum level of calcium in apple fruit maintain fruit flesh firmness and reduces the incidence of physiological disorders such as water core, bitter pit and internal breakdown and postharvest decay (Conway et al., 2002). Soil treatments with calcium to increase fruit calcium content have often met with very little success but direct application of calcium to the fruit is the most effective method for increasing fruit calcium content, accomplished by pre-harvest sprays or postharvest dips or vacuum or pressure infiltration (Conway et al., 2002). According to Martin et al. (1960), magnesium nitrate spray increased the incidence of bitter pit but calcium nitrate decreased it. The optimum level of Ca content may be different for various cultivars (Johnson, 1980; Dris et al., 1998). The present experiment was, therefore, conducted to evaluate the influence of CaCl2 concentration and dipping duration on internal quality changes in apple cultivars. Thus this experiment was conducted to optimize the CaCl2 concentration and dipping duration to minimize the quality losses during storage in apple cultivars.

6.2. MATERIALS AND METHODS

The influence of CaCl2 treatment on storage performance of apple cultivars was investigated at Postharvest Laboratory, Khyber Pakhtunkhwa Agricultural University, Peshawar-Pakistan during 2009-10. The fruits from three apple cultivar Royal Gala, Mondial Gala and Golden Delicious were harvested at commercial pale yellow maturity stage. The fruit were harvested from 10 different plants. The harvested fruits were checked from mechanical injury or symptoms of diseases and physiological disorders. Healthy and sound fruits of uniform size were selected and were divided into two groups each containing 150 fruits. One group of fruits dipped in distilled water for 12 minutes and the other was treated with 9% CaCl2 solution by dipping for the same duration. Calcium solution was made from analytical grade Calcium chloride. The fruit understudy were carefully placed in plastic buckets and slight pressed with wooden top made according to the size of the bucket. The surface moisture was removed with gentle air blower. The fruits were then shifted to cold storage at 5±1oC with 60-70% relative humidity for a period of 150 days. The calcium content of apple fruits were determined as described in experiment No. 2. The experiment was laid out in completely randomized design (CRD) with twelve treatment combinations repeated three times. The detail of the post-harvest experiment is as under Cultivars (C) = (Royal Gala, Mondial Gala and Golden Delicious) Storage duration (S) = (0 and 150 days)

CaCl2 conc. (Ca) = (0 and 9%) The data were recorded and statistically analyzed for the following post harvest quality parameters as described in experiment No. 1 at storage of 0 and 150 days. Weight loss (%), Percent juice content, Starch content, Total soluble solids (0Brix) Total sugars, Percent acidity, TSS/Acid ratio, Ascorbic acid (mg/100g), Fruit flesh firmness (kg/cm2), Density of fruit, Bitter pit (%) and Soft rot (%)

Statistical Procedures The data calculated on different parameters were subjected to Analysis of Variance (ANOVA) technique to observe the differences between the different treatment as well as their interactions. In cases where the differences were significant, the means were further assessed for differences through Least Significant Difference (LSD) test. Statistical computer software, MSTATC (Michigan State University, USA), was applied for computing both the ANOVA and LSD.

6.3. RESULTS

Weight loss (%) The data on percent weight loss indicated that there were significant variations among cultivars, storage duration and CaCl2 application. The interaction of cultivar × storage and storage × CaCl2 application was also significant (Table 6.1). The data presented in Table 6.1 indicates that there were significant differences in weight loss of different apple cultivars. The maximum weight lost (2.11%) was recorded in cultivar Golden Delicious which was significantly higher than Royal Gala (1.61%), and Mondial Gala (1.55%), with the difference in weight loss in the later two cultivars being non significant. The mean percent weight loss increased significantly with storage for 150 days to the maximum of 3.52% but a significant decreased was recorded with CaCl2 application (1.16%) as compared to 2.36% in control. The interaction of cultivars and storage duration was also significant. The highest weight loss (4.23%) was recorded in cultivar Golden Delicious after 150 days storage, followed by Royal Gala and Mondial Gala with weight loss of 3.22 and 3.10% respectively. However, the difference in the later two varieties was non significant

(Figure 6.1). The interaction effect of storage and CaCl2 application was significant.

The greatest weight loss (4.71%) recorded in fruit treated with 9% CaCl2 at 0 day storage, followed by apple fruits with 2.32% treated with same application after 150 days storage (Figure 6.2).

Juice content (%) The data regarding percent juice content revealed that there were significant differences among cultivars, storage duration and CaCl2 application. The interaction effect was non significant (Table 6.1). Significant variations were observed in juice content of different apple cultivars with the maximum juice content (59.20%) recorded in Royal Gala. However, the minimum juice content (49.17%) was recorded in Golden Delicious, followed by Mondial Gala with 57.65% juice content (Table 6.1). The juice content significantly decreased from 63.44% for fresh harvest fruit to 47.23% for fruits stored for 150 days but was significantly high with CaCl2 application (57.58%) as compared to 53.10% recorded in control. Loss of starch content (Score)

The data pertaining to starch score revealed no significant difference among apple cultivars but it declined significantly with storage and remained with increasing CaCl2 application. The interactions of storage × CaCl2 also significantly affected the starch score but storage × cutivar, cutivar × CaCl2 and cultivar × storage × CaCl2 were non significant (Table 6.1). The starch content score decreased significantly during storage from 6.95 for fresh harvested fruit to 3.23 for fruit stored for 150 days. The application of CaCl2 resulted in significantly high starch score 5.49 as compared to 4.69 in control. The interaction effect of storage and CaCl2 application was also significant. The maximum starch score (7.00) recorded in untreated fruits 0 day storage, followed by apple fruits with starch score (6.90) treated with 9% CaCl2 application after 150 days storage, however these two starch scores was at par with each other. The minimum starch score (2.38) observed in untreated fruits after 150 days storage (Figure 6.3).

Total soluble solids The data on total soluble solids (TSS) revealed no significant differences among cultivars, whereas, the effect of storage and CaCl2 application of dipping solution was significant. The interaction effect of storage × CaCl2 was non significant (Table 6.1). The total soluble solids contents of the fruit increased significantly during storage from 9.91 in fresh harvested fruit to 12.46% in fruit stored for 150 days but CaCl2 application decreased the total soluble solids to 10.92% in contrast to 11.44% in control. The interaction effect of storage and CaCl2 application was also significant. The maximum total soluble solids (13.00%) recorded in un treated fruits after 150 days storage, followed by apple fruits with total soluble solids (11.91%) in fruits treated with CaCl2 solution, however these two values was at par with each other. The minimum total soluble solids (9.88%) observed in untreated fruits at 0 day storage (Figure 6.4).

Total sugar (%) The data on total sugar revealed no significant differences among cultivars, whereas, the effect of storage and CaCl2 application was significant (Table 6.1). The interaction effect was of storage and CaCl2 application was also significant. The total sugar significantly increased during storage from 9.41% for fresh harvested fruit to 11.96% with 150 days storage but were significantly high (10.87%) in non

treated apple fruits as compared to 10.50 % with CaCl2 application. The interaction effect of storage and CaCl2 application was also significant. The maximum total sugar (12.36%) recorded in untreated fruits at 150 days storage, followed by apple fruits with total sugar (11.56 %) in fruits treated with CaCl2 solution after 150 days storage. The minimum total soluble solids (9.39%) observed in untreated fruits at 0 day storage (Figure 6.5).

Titratable acidity (%) The data in Table 6.1 indicate that there were significant differences in titratable acidity among cultivars, storage durations and CaCl2 application as well as the interaction of cultivars × storage and storage × CaCl2 application. The apple cultivars showed significant variations in percent titratable acidity with the maximum titratable acidity (0.54%) recorded in Mondial Gala and followed by Royal Gala with 0.52%. However, the minimum titratable acidity (0.48%) recorded in Golden Delicious. The percent acidity significantly decreased from 0.64%, recorded in fresh harvested fruits to 0.39% in apple fruit stored for 150 days. The titratable acidity increased significantly from 0.49% for non treated apple fruits to 0.53% with

CaCl2 solution. Titratable acidity of apple juice was significantly affected by the interaction of apple cultivars and storage durations. The maximum titratable acidity (0.69%) was recorded in cultivar Mondial Gala at 0 day storage, followed by Royal Gala with weight loss of 0.65%, with the difference being non significant. The minimum titratable acidity (0.38%) was observed in cultivars Mondial Gala after 150 days storage, followed by Golden Delicious and Royal Gala with titratable acidity of 0.39 and 0.40% respectively, however, the variation was non significant among these three cultivars (Figure 6.6). The interaction effect of storage and CaCl2 application was also significant. The maximum titratable acidity (0.64%) recorded in un treated fruits at 0 day storage, followed by apple fruits with 0.63% in fruits treated with

CaCl2 solution, however these two values was at par with each other. The minimum titratable acidity (0.35%) observed in untreated fruits at 0 day storage (Figure 6.7).

TSS/Acid ratio The data in Table 6.1 indicated significant differences in TSS/Acid ratio in different storage durations and CaCl2 application of dipping solution. The interaction effect of storage and CaCl2 application was also significant.

Storage had a significant effect on TSS/Acid ratio of apple juice. The TSS/Acid ratio increased from 15.75 in fresh fruit (S0) to 32.63 with storage for 150 days. The apple fruits dipped in CaCl2 solution also had lower TSS/acid ratio of 21.69 as compared to

26.69 recorded for control treatment. The interaction effect of storage and CaCl2 application was also significant. The maximum TSS/Acid ratio (37.75) recorded in untreated fruits after 150 days storage, followed by apple fruits with 27.51 in fruits treated with CaCl2 solution, however these two values was at par with each other. The minimum TSS/Acid ratio (15.63) observed in untreated fruits at 0 day storage (Figure 6.8).

Table 6.1. The effect of cultivars, storage and CaCl2 application on weight lost, percent juice, starch, TSS (%), total sugar (%), titratable acidity (%), and TSS/Acid ratio of apple cultivars

Cultivars Weight Juice Starch TSS Total Titratable TSS/Acid loss content (Score) (%) sugar acidity ratio (%) (%) (%) (%) Royal Gala 1.61 b 59.20 a 5.12 11.08 10.59 0.52 ab 23.45 Mondial Gala 1.55 b 57.65 ab 4.97 11.02 10.63 0.54 a 23.42 Golden Delicious 2.11 a 49.17 b 5.18 11.45 10.84 0.48 b 25.71 LSD value 0.33 9.40 ns ns ns 0.06 ns Storage (days) 0 0.00 63.44 6.95 9.91 9.41 0.64 15.75 150 3.52 47.23 3.23 12.46 11.96 0.39 32.63

Significance level * * * * * * *

CaCl2 (%) 0 2.36 53.10 4.69 11.44 10.87 0.49 26.69 9 1.16 57.58 5.49 10.92 10.50 0.53 21.69 Significance level * * * * * * * Interactions Significance Level C × S * ns ns ns ns * ns C × Ca ns ns ns ns ns ns ns S × Ca * ns * * * * * C × S × Ca ns ns ns ns ns ns ns

Mean followed by similar letter(s) in column do not differ significantly ns = Non Significant * = Significant at 5% level of probability. C × S = Interaction of cultivar and storage duration C × Ca = Interaction of cultivar and CaCl2 concentration S × Ca = Interaction of storage duration and CaCl2 concentration C × S × Ca = Interaction of cultivar, storage duration and CaCl2 concentration

5 4.5 4 3.5 3 2.5 2

Weight Loss (%) Loss Weight 1.5 1 0.5 0 Royal gala Mondial gala Golden delicious Cultivars

Figure 6.1 Variation in weight loss (%) in apple fruits stored for 150 days of different apple cultivars

7

6

5

4

3

Weight Loss (%) Loss Weight 2

1

0 0 9 CaCl2 (%)

Figure 6.2 Effect of CaCl2 concentrations on weight loss (%) in apple fruits stored for 150 days

10 9 8 7 6 0

5 150

4 Starch (Scores) Starch 3 2 1 0 0 9

CaCl2 (%)

Figure 6.3 Interaction effect of CaCl2 application and storage duration on starch scores of apple

18 16 14 12 0 10 8 150 6

Total Soluble Solids (%) SolubleSolids Total 4 2 0 0 9 CaCl2 (%)

Figure 6.4 Interaction effect of CaCl2 application and storage duration on TSS of apple

16 14 12

10 0 8 150

6 Total Sugars Sugars (%) Total 4 2 0 0 9 CaCl2 (%)

Figure 6.5 Interaction effect of CaCl2 application and storage duration on total sugar (%) of apple

0.8 0.7 0.6 0.5 0

0.4 150 0.3

Titratable Acidity (%) Acidity Titratable 0.2 0.1 0 Royal gala Mondial gala Golden delicious Cultivars

Figure 6.6 Interaction effect of cultivar and storage duration on titratable acidity (%) of apple

0.9 0.8 0.7 0.6 0.5 0 0.4 150 0.3

Titratable Acidity (%) Acidity Titratable 0.2 0.1 0 0 9 CaCl2 (%)

Figure 6.7 Interaction effect of CaCl2 application and storage duration on titratable acidity (%) of apple

60

50

40 0 30 150

20 TSS/Acid Ratio TSS/Acid 10

0 0 9

CaCl2 (%)

Figure 6.8 Interaction effect of CaCl2 application and storage duration on TSS/Acid ratio of apple

Ascorbic acid (mg/100g) The data on ascorbic acid as shown in Table 6.2 indicated that ascorbic acid content of apple fruit was significantly different among different cultivars, storage duration and CaCl2 application as well as the interaction of cultivar × storage and storage ×

CaCl2 applications. The maximum ascorbic acid content was recorded in cultivar Royal Gala (12.88 mg/100g) followed by Mondial Gala with 12.73 mg/100g, however, these two cultivars are at par with each other. The least ascorbic acid was observed in cultivar Golden Delicious (10.79 mg/100g) (Table 6.2). The ascorbic acid decreased significantly from 14.16 observed in fresh fruits to 10.11 mg/100g after 150 days storage at 5±1oC. The ascorbic acid was significantly high (12.97 mg/100g) recorded in non treated apple fruits to 11.30 mg/100g in fruits dipped CaCl2 solution. The interaction effect of cultivars and storage durations on ascorbic acid was also significant. The maximum ascorbic acid (15.12 mg/100g) was recorded in cultivar Mondial Gala, followed by Royal Gala with 15.03 mg/100g after 0 day storage, however, these two cultivars was at par with each other. The minimum ascorbic acid (9.26 mg/100g) was observed with 150 days storage in cultivar Golden Delicious, followed by Mondial Gala and Royal Gala with ascorbic acid content of 1.35 and 10.73 mg/100g respectively. However, the differences among all the three cultivars was statistically non significant after 150 days storage (Figure 6.9). The interaction effect of storage and CaCl2 application was also significant. The maximum ascorbic acid (14.26 mg/100g) recorded in fruits dipped in 9% CaCl2 solution at 0 day storage while, the minimum ascorbic acid (8.55 mg/100g) observed in untreated fruits at 0 day storage (Figure 6.10). Fruit flesh firmness (kg/cm2) It is clear from the table 6.2 that fruit flesh firmness was significantly varied among cultivars, storage durations and CaCl2 application of dipping solution as well as the interaction of cultivar × storage and storage × CaCl2 applications. There was significant difference in fruit flesh firmness among apple cultivars. The maximum fruit flesh firmness of 5.46 kg/cm2 was observed in Royal Gala, followed by Mondial Gala with the fruit flesh firmness of 5.43 kg/cm2, however the difference in these cultivars was non significant. The minimum fruit flesh firmness (4.87 kg/cm2) was recorded in Golden Delicious. The fruit flesh firmness significantly decreased from 6.34 kg/cm2 for fresh fruits to 4.17 kg/cm2 for fruits after 150 days storage

(Table 6.2) but significantly increased from 4.89 kg/cm2 recorded in non treated apple 2 fruits to 5.62 kg/cm in fruits dipped in 9% CaCl2 solution. The interaction effect of cultivars and storage durations on fruit flesh firmness was also significant. The maximum fruit flesh firmness (6.47 kg/cm2) was recorded in cultivar Royal Gala, followed by Mondial Gala and Golden Delicious with 6.42 and 6.13 kg/cm2 respectively after 0 day storage, however, these three cultivars was at par with each other. The minimum fruit flesh firmness (3.61 kg/cm2) was observed with 150 days storage in cultivar Golden Delicious (Figure 6.11). The interaction effect of storage and CaCl2 application was also significant. The maximum fruit flesh firmness (6.45 2 kg/cm ) recorded in fruits dipped in 9% CaCl2 solution at 0 day storage while, the minimum fruit flesh firmness (3.55 kg/cm2) observed in untreated fruits at 0 day storage (Figure 6.12). Density of fruit (g/cm3) The data regarding density of apple fruit in relation to different storage duration and

CaCl2 application of dipping whereas, the interaction effect was non significant (Table 6.2). A significant decrease in density of fruit was recorded with storage duration of 150 days. The density of fruit decreased from 0.79 g/cm3 recorded in fresh harvested fruit 3 to 0.74 g/cm observed in fruit stored for 150 days. The application of CaCl2 solution retained significantly higher fruit density (0.78 g/cm3) as compared to non treated apple fruits (0.76 g/cm3). Bitter pit (%) There was significant variation in bitter pit incidence among different apple cultivars, storage durations and CaCl2 application of dipping solution as well as the interaction of cultivar × storage, cultivar × CaCl2 application, storage × CaCl2 applications of dipping solution and cultivar × storage × CaCl2 application of dipping solution (Table 6.2). The apple cultivars varied significantly in bitter pit incidence in apple fruits. The lowest bitter pit incidence (2.92%) was measured in Royal Gala, followed by Mondial Gala with 3.34% of bitter pit incidence, with the difference being non significant. The highest incidence of bitter pit (6.96%) was observed in cultivar Golden Delicious. The bitter bit incidence was 0% in fresh harvested fruits but increased to 8.81% in fruit stored for 150. The bitter pit incidence in non treated apple fruits (7.78%) significantly decreased to 1.03% in fruits dipped in CaCl2 solution. The bitter pit

incidence increased significantly with increasing storage duration in all the cultivars under study. After 150 days storage, the bitter pit incidence was significantly lower in Royal Gala and Mondial Gala with 5.84 and 6.67% respectively. The maximum bitter pit incidence of 13.93% was recorded in cultivar Golden Delicious (Figure 6.13). The interaction of cultivar and CaCl2 application also significantly affected the bitter pit incidence. The maximum bitter pit incidence (12.10%) observed in untreated fruits of cultivar Golden Delicious. The minimum bitter pit incidence of 0.60% recorded in cultivar Royal Gala treated with CaCl2 solution (Figure 6.14). The interaction of storage and CaCl2 application significantly affected the incidence of bitter pit. The maximum incidence of bitter pit (15.56%) observed in fresh fruits treated with 9%

CaCl2 solution, followed by 2.06% in fruits treated with the same application after

150 days storage (Figure 6.15). The interaction of cultivar, storage and CaCl2 solution also significantly affected the incidence of bitter pit incidence. The maximum bitter pit incidence (24.20%) recorded in untreated fruits of cultivar Golden Delicious after 150 days storage which was significantly decreased to 3.65% after treated with 9%

CaCl2 solution (Figure 6.16). Soft rot (%)

The data in Table 6.2 revealed that the storage durations and CaCl2 application of dipping solution significantly influenced the percent soft rot of apple fruits, however, the variation was non significant among cultivars. The interaction of cultivar × storage and storage durations × CaCl2 application of dipping solution for soft rot of fruit was also significant. The soft rot in apple fruit increased with increase in storage durations, it increased to 10.57% during 150 days storage. The soft rot incidence significantly decreased from

9.16% recorded in non treated apple fruits to 1.41% in fruits dipped in 9% CaCl2 solution. The interaction of cultivars and storage duration showed significantly high soft rot incidence with increasing storage duration. After 150 days storage, the soft rot incidence was the maximum (12.54%) in cultivar Golden Delicious followed by Royal Gala and Mondial Gala with 9.70 and 9.47% respectively, however, all the three cultivars were at par with each other (Figure 6.17). The interaction of storage and CaCl2 application significantly affected the incidence of soft rot. The maximum incidence of soft rot (18.32%) observed in fresh fruits treated with 9% CaCl2 solution, followed by 2.82% in fruits treated with the same application after 150 days storage (Figure 6.18).

Table 6.2. The effect of calcium application and storage on ascorbic acid (mg/100g), fruit flesh firmness (kg/cm2), fruit density (g/cm3), bitter pit (%) and soft rot (%) of apple cultivars

Cultivars Ascorbic acid Fruit flesh Fruit Bitter pit Soft rot (mg/100g) firmness density (%) (%) (kg/cm2) (g/cm3)

Royal Gala 12.88 a 5.46 a 0.78 2.92 b 4.85 Mondial Gala 12.73 a 5.43 a 0.79 3.34 b 4.74 Golden Delicious 10.79 b 4.87 b 0.73 6.96 a 6.27 LSD value 1.10 0.43 ns 1.13 ns Storage (days) 0 14.16 6.34 0.79 0.00 0.00 150 10.11 4.17 0.74 8.81 10.57 Significance level * * * * * CaCl (%) 2 0 11.30 4.89 0.76 7.78 9.16 9 12.97 5.62 0.78 1.03 1.41 Significance level * * * * *

Interactions Significance Level

C × S * * ns * *

C × Ca ns ns ns * ns

S × Ca * * ns * *

C × S × Ca ns ns ns * ns

Mean followed by similar letter(s) in column do not differ significantly ns = Non Significant * = Significant at 5 % level of probability C × S = Interaction of cultivar and storage duration C × Ca = Interaction of cultivar and CaCl2 concentration S × Ca = Interaction of storage duration and CaCl2 concentration C × S × Ca = Interaction of cultivar, storage duration and CaCl2 concentration

18 16 14 12 0 10 150 8 6

4 Ascorbic Acid Acid (mg/100g) Ascorbic 2 0 Royal gala Mondial gala Golden delicious Cultivars

Figure 6.9 Interaction effect of cultivar and storage on ascorbic acid (mg/100g) of apple

20 18 16 14 12 0 10 150 8 6

Ascorbic Acid Acid (mg/100g) Ascorbic 4 2 0 0 9

CaCl2 (%)

Figure 6.10 Interaction effect of CaCl2 application and storage duration on ascorbic acid (mg/100g) of apple

8

7 ) 2 6 0 5 150 4

3 Firmness (kg/cm Firmness 2 1 0 Royal gala Mondial gala Golden delicious

Cultivars

Figure 6.11 Interaction effect of cultivar and storage duration on fruit flesh firmness (kg/cm2) of apple fruit

9

8 )

2 7 6 0 5 4 150

3 Firmness (kg/cm Firmness 2 1 0 0 9 CaCl2 (%)

Figure 6.12 Interaction effect of CaCl2 application and storage duration on fruit flesh firmness (kg/cm2) of apple

18 16 14 12 10 8

Bitter Pit (%) Pit Bitter 6 4 2 0 Royal gala Mondial gala Golden delicious Cultivars

Figure 6.13 Variation in bitter pit (%) incidence in apple fruits after 150 days storage of different cultivars

16 14 12

10 0 8 9 6

Bitter Pit (%) Pit Bitter 4 2 0 Royal gala Mondial gala Golden delicious

Cultivars

Figure 6.14 Interaction effect of cultivar and storage on bitter pit (%) incidence in apple

25

20

15

10 Bitter Pit (%) Pit Bitter 5

0 0 9 CaCl2 (%)

Figure 6.15 Effect of CaCl2 concentrations on bitter pit (%) incidence in apple fruits after 150 days storage

30

25

20 0

15 9

10 Bitter Pit (%) Pit Bitter

5

0 Royal gala Mondial gala Golden delicious Cultivars

Figure 6.16 Interaction effect of cultivar and storage on bitter pit (%) of apple cultivar stored for 150 days

20 18 16 14 12 10 8

Soft Rot (%) Rot Soft 6 4 2 0 Royal gala Mondial gala Golden delicious Cultivars

Figure 6.17 Influence of 150 days storage on soft rot (%) incidence in apple cultivars

35

30

25

20

15 Soft Rot (%) Rot Soft 10

5

0 0 9 CaCl2 (%)

Figure 6.18 Effect of CaCl2 concentrations on soft rot (%) incidence in apple fruits stored for 150 days Calcium content of the fruit The data presented in Table 6.3 revealed that apple cultivar varied significantly in calcium content of the fruit. The calcium content in different cultivars ranged from 38 mg kg-1 in Golden delicious to 44 mg kg-1 in Royal Gala. The Calcium concentration

-1 of fruits dipped in 0% CaCl2 (control fruit) increased to 121, 105 and 105 mg kg in cultivars Royal Gala, Mondial Gala and Golden respectively.

Table 6.3. Effect of CaCl2 dipping on calcium content (mg/kg) of apple cultivars

Cultivar CaCl2 concentration (%) 0 9 Royal Gala 44 121 Mondial Gala 41 115 Golden Delicious 38 105

6.4. DISCUSSION

Percent weight loss and Juice content (%) The moisture content of fruits is an important quality criterian (Gorini et al., 1979) and the loss of turgor pressure and subsequent softening is depends on moisture loss (Vander-Beng, 1981). The weight loss depends on water present in the fruit and the structure of the skin and nature of waxes on the surface (Babos et al., 1984; Veravrbeke et al., 2003). Considerable variation has been observed in the skin thickness of different apple cultivars and even the same cultivar may show significant variation with in different years of production (Homutova and Blazek, 2006). The data on weight loss of different apple cultivars indicated that the maximum weight loss in cultivar Golden Delicious was 26.54% higher than Mondial Gala, though the difference in Mondial Gala and Royal Gala non significant. Storage for 150 days resulted in 3.52% mean percent weight loss which decreased significantly 50.85% with CaCl2 application as compared to untreated control (Table 6.1). The weight loss in fruits increased linearly with increase in storage duration due to water loss and respiration (Blampired, 1981; El-Shennawi, 1989; Gavlheiro et al., 2003; Erturk, 2003; Ghafir et al., 2009). The weight loss decreased significantly with increase in

CaCl2 concentration or dipping solution in CaCl2 solution (Table 6.1). The decrease in weight loss with increase in CaCl2 concentration or dipping duration is in accordance with Ashore (2000) and Hayat et al. (2003). The juice content of apple fruit depends mainly on the water content of the fruit and decreasing the rate of water loss increases the juice content (Dzonova et al., 1970; Tu et al., 2000). Thus, cultivar Golden Delicious, characterized by more weight loss had 16.94% lower juice content (Dzonova et al., 1970) as compared to Royal Gala. The decrease in percent juice decline is due to the water loss from the tissue which increases with storage duration

(Allan et al., 2003). The CaCl2 application resulted in significantly high juice content which was 8.10% more than the untreated control (Table 6.1).

Loss of starch content (Score) While the starch content score was non significant among different apple cultivars, it decreased by 53.53% during storage for 150 days. The decline in starch content was retarded by the application of CaCl2 which resulted in 14.57% lower loss of starch content than the non treated control fruits (Table 6.1). The starch is the major storage

carbohydrates in apple fruit (Beaudry et al., 1989), which is converted to sugars at the onset of ripening and during storage to meet the respiratory demand of the fruit (Bidabe et al., 1970; Crouch, 2003), thus it is likely to observe the decline in starch content during storage of apple fruit. The apple fruit converted the stored starch to free sugars in order to meet the respiratory demands. Since calcium application delay the ripening process of fruit, therefore the conversion of starch to sugar is also less with CaCl2 application (Kadir, 2005).

Titratable acidity (%) The apple cultivars had significantly affected percent titratable acidity with the maximum titratable acidity recorded in Royal Gala and Mondial Gala was 7.69 and 11.11% higher than Golden Delicious respectively. The percent acidity decreased by 39.06% with storage for 150 days in contrast to fresh harvested fruits. The decrease in titratable acidity increased significantly with CaCl2 treatment, it was 7.54% higher than the non treated apple fruits (Table 6.1). The decline in titratable acidity depends on the rate of metabolism (Murata and Minamide, 1970; Clarke et al., 2001) especially respiration which consumed organic acid and thus decline acidity (Rivera, 2005; Ghafir et al., 2009). The decrease in acidity also results due to the utilization of organic acids, raw materials for synthesis of other compounds during ripening. The organic acids are consumed in respiration, resulting in lower acidity with increasing storage duration (Rivera, 2005; Ghafir et al., 2009).

Total soluble solids The total soluble solids contents of the fruit followed similar pattern to the total sugars. While the apple cultivars understudy had no significant difference in TSS, it increased significantly during storage so that it was 20.46% higher in fruit stored for 150 days as compared to the fresh harvested fruits stored for 0 days (Table 6.1). The increase in TSS was decreased by 4.55% when the fruit were treated with CaCl2 as compared to non treated control (Table 6.1). The increase in TSS may be due to the enzymatic conversion of higher polysaccharides such as starches to simple sugars during ripening (Hussain et al., 2008). The less increase in TSS with increasing CaCl2 concentrations may be due to the delay in natural physiological processes like ripening and senescence by CaCl2, due to the inhibition or retardation of conversion to simple sugars from complex polysacchrides (Izumi and Watada 1994; Agar et al.,

1999; Rosen and Kader, 1989; Kader, 1986). It is however, also important that the loss of moisture may concentrate the cell sap, resulting in low juice content but high TSS percentage.

Total sugar (%) The sugars content of apple fruit contribute to the fruit sweetness, and, thus, is a major fruit quality characteristics. The total sugars were non significantly different among different apple cultivars but apple fruit stored for 150 days had 21.32% high total sugars than freshly harvested fruits (Table 6.1). In apple fruit the sugars tend to increase with maturation (Wani et al., 2008). The apple fruit accumulate starch at the early stages of maturation, which is hydrolyzed to sugars at edible maturity (Magein and Leurquin, 2000) and during storage (Beaudry et al., 1989), resulting in increased total sugars with increased storage duration (Bidabe et al., 1970; Crouch, 2003). The increase in sugars during storage is therefore in line with the observation on loss of starch during the storage period. The calcium treatment of the apple fruit retarded the trend of increasing total sugars so that there was 3.4% less total sugars as compared to the untreated fruits.

TSS/Acid ratio The different apple cultivars under study were statistically at par in TSS/Acid ratio which increased by 107.17% during 150 days storage. The CaCl2 treatment of apple fruit decreased the trend of increasing TSS/Acid ratio. Thus, the TSS/Acid ratio in control fruit was 18.73% higher than the fruits treated with CaCl2 solution (Table 6.1).

Since CaCl2 application caused significant decrease in TSS and increase in titratable acidity, it is reasonable to observe lower TSS/acid ratio with CaCl2 application. The TSS/ Acid ratio of apple and other fruits is a major quality parameter (Weibel et al., 2004; Peck et al., 2006). The TSS/ Acid ratio generally increases with increasing storage duration. The increase in TSS/Acid ratio can be attributed to starch breakdown resulting in free sugars (Beaudry et al., 1989) and decline in organic acids due to its consumption in respiration (Mahajan, 1994; Rivera, 2005; Ghafir et al., 2009).

Ascorbic acid (mg/100g)

Ascorbic acid is usually considered as an index of nutrient quality in apple fruit. Ascorbic acid is a bioactive compound having antioxidant properties (Lata, 2007). The ascorbic acid decreased significantly with storage (Purvis, 1983; Hayat et al., 2003). There was no significant different in ascorbic acid content of Royal Gala and Mondial Gala but the maximum ascorbic acid recorded in cultivar Royal Gala was 16.23% higher than cultivar Golden Delicious (Table 6.2). The apple cultivars differ significantly in their ascorbic acid content (Davey et al., 2007). For example apple cultivars Mushhadi, Amri and Kalakulu had significantly higher vitamin C than Kalakulu (Ali et al., 2004; Nour et al., 2010) which is about 12.8 mg/100 g fruit. According to Lee et al. (2003) the ascorbic acid decreased significantly during storage for 150 by 28.60% as compared to fresh fruits (Table 6.2). The decrease in ascorbic acid was 12.97% less in CaCl2 treated fruits as compared to non treated control (Table 6.2). The ascorbic acid is a high labile vitamin which tends to decline during storage (Adisa, 1986). The ascorbic acid loss during storage is known to be due to its antioxidant activity especially under postharvest storage conditions (Davey et al., 2000). The ascorbic acid can be irreversible oxidized (Parviainen and Nyyssonen, 1992; Pardio-Sedas et al., 1994), thus causing a decrease during storage (Jung and

Watkins, 2008). The retention of relatively high ascorbic acid with increasing CaCl2 concentrations or duration may due to the regulation of oxidative processes is the cytosol leading to ascorbic acid degradation (Faust and Shear, 1972).

Fruit flesh firmness (kg/cm2) Fruit flesh fruit flesh firmness is an important criterion for edible quality and market value of apples (Stow, 1995; De-Ell et al., 2001; Weibel et al., 2004; Peck et al., 2006) and loss of fruit flesh firmness is a serious problems resulting in quality losses (Kov et al., 2005). The apple cultivars varied significantly in fruit flesh firmness was recorded among apple cultivars. Cultivar Royal Gala had the maximum fruit flesh firmness, which was at par with Mondial Gala but 10.81% higher than Golden Delicious. The difference in fruit flesh firmness of different apple cultivars is attributed to differences in pectin composition of different cultivars (Billy et al., 2008). The fruit flesh firmness significantly decreased by 34.70% with 150 days storage and this trend was retarded by the treatment with CaCl2 solution so that the fruit flesh firmness 12.99% higher as compared to the untreated control. The fruit flesh firmness of the apple fruit significantly decreased with increasing storage (Table

6.2). The fruit flesh firmness of the apple fruit is due to texture of the flesh and textural changes of fruits especially the cell wall breakdown (Fuller, 2008) due to enzymatic activities (Yamaki and Matsuda, 1977) and pectin solubalization (Bartley et al., 1982; Jackman and Stanley, 1995; Chang-Hai et al., 2006), reducing the mechanical strength of cell walls which decrease the fruit flesh firmness in apple fruits (Kov et al., 2003; Kov et al., 2005). The retention of fruit flesh firmness with increasing calcium concentration or dipping duration can be attributed to the formation of calcium pectates leading to increased rigidity of the cell wall and, thus, improved turgor pressure (Jackman and Stanley 1995; Luna-Guzman and Barrett, 2000).

Fruit density (g/cm3) The density of the fruit is a physical characteristic that has been can as maturity (Wolfe et al., 1974) and quality index. In Potato, the tuber‟s density is generally correlated with the starch content of tubers (Zaltzman et al., 1987), dry matter (Wilson and Lindsay, 1969), as well as the mechanical resistance of tubers (Hudson, 1975). The density of the fruit may be influenced by rind thickness (Cohen, 1972), juice content (Zaltzman et al., 1987), dry matter (Jordan et al., 2000) total sugars and starch contents (Robert et al., 2000). Thus, fruit density also been used as maturity and quality index in many fruits and vegetables such as apricots, strawberries, tomato, pea, etc (Wolfe et al., 1974; Zaltzman et al., 1987; McGlone et al., 2007). No significant decrease in density of fruit was recorded among different apple cultivars under study but the storage for 150 days decline fruit density by 6.33%. The treatment with CaCl2 solution retained significantly higher fruit density. The fruit density in

CaCl2 treated fruits was 2.56% higher the non treated control (Table 6.2). The changes in density of apple fruit is a function air spaces and solutes dissolved in the cell sap (Marlow and Loescher, 1984; Bayindirli, 1993). Therefore, the specific gravity of apple fruit from different cultivars could be significantly different (Vincent, 1989) due to differences in biochemical composition (Homutova and Blazek, 2006; Ghafir et al., 2009) and moisture loss during storage (Rivera, 2005). Therefore, the density of apple fruit is high in fresh fruits and decline during storage (Sakiyama and Nakamura, 1976) due to collapse of intercellular spaces and loss of moisture (Mitropoulos and Lambrinos, 2000). Both increasing the concentration of Calcium in dipping solution or duration of dipping in calcium solution decreased the loss of density in apple fruit. The calcium regulates the fruit flesh firmness by maintaining

cell wall structure (Poovaiah, 1986; Conway and Sams, 1987) and delaying the natural ripening and senescence (Rosen and Kader, 1989). Thus, the calcium may decrease the development of air spaces associated with ripening and responsible for the loss of specific gravity (Ruess and StoÈsser, 1993).

Bitter pit (%) Bitter pit is a physiological disorder, characterized by depressed brown lesions in the skin of the fruit (Ferguson and Watkins, 1989). The apple cultivars varied significantly in bitter pit incidence on fruits. Cultivar Royal Gala has the lowest bitter pit incidence (2.92%) which was statistically at par with Mondial Gala but significantly lower than cultivar Golden Delicious, which had 58.05% higher bitter pit incidence than Royal Gala (Table 6.2). Crouch (2003) reported that the incidence of bitter pit depends on genetic factors and apple cultivar Red Delicious is more susceptible to bitter pit as compared to Golden Delicious. The bitter bit incidence increased by 8.81% in fruit stored for 150 days but decreased significantly (11.32%) with CaCl2 treatment. The incidence of bitter pit generally increased with increasing storage duration (Pesis et al., 2009). The lower bitter pit incidence with CaCl2 treatment is in accordance with earlier reports that Ca treatments can decrease bitter pit incidence. The incidence of bitter pit is related to plant nutrition (Fallahi et al., 1997) especially Ca concentration (Crouch, 2003; Pesis et al., 2009) and can be decreased by pre (Peryea et al., 2003) and postharvest Ca application (Reid and Padfield, 1975; Jones and Higgs, 1982).

Soft rot (%) The soft rot in apple fruit increased with increase in storage durations, it increased to

10.57% during 150 days storage but decreased significantly with dipped in CaCl2 solution (Table 6.2). The bitter pit incidence on apple fruit depends on cultivar but generally increase during storage (Kader, 1985; Spotts et al., 1999). The incidence of soft rot was 84.61% more in untreated control as compared to the fruit treated with

CaCl2. The decreased soft rot incidence with increased calcium concentration may be due to the calcium-induced delay in natural ripening and senescence (Agar et al., 1999), which causes the fruit susceptible to pathogens.

Calcium content of the fruit

Dipping of apple fruit in 9% CaCl2 solution for 9 minutes resulted in 2.75 to 2.80 fold increase in the calcium content of apple fruit. It indicates that dipping in calcium chloride solution is an effective way of increasing the calcium content of the fruit.

Furthermore, apple cultivars had comparable incorporation of CaCl2. Since the CaCl2 uptake is mainly through the lenticels (Dewey and Lee, 1980), it is likely to observe about similar increase in calcium content of all cultivars under study.

6.5. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary

The research on “Influence of CaCl2 treatment on storage performance of apple cultivars” was carried out at Horticulture Postharvest Laboratory, Khyber Pakhtunkhwa Agricultural University Peshawar Pakistan during 2009-10. The fruits were harvested from apple cultivars: Royal Gala, Mondial Gala and Golden Delicious at commercial maturity stage at Matta, Swat. The fruits were treated with either 0 or

9% CaCl2 solution for the period of 12 minutes and stored for the period of 150 days at 5±1oC with 60-70% relative humidity. The data was recorded for all the parameters at 0 and 150 days storage. The storage performance of apple cultivars such as juice content, starch score, total sugar, total soluble solids, TSS/Acid ratio, ascorbic acid, fruit flesh firmness and density of fruit was significantly affected by calcium treatment. The highest juice content, ascorbic acid, fruit flesh firmness and least bitter pit incidence were recorded in cultivar Royal Gala, whereas Mondial Gala has the least weight loss and maximum titratable acidity. The apple cultivar Golden Delicious was more susceptible to the bitter pit incidence during storage. CaCl2 application had significantly lower the percent weight loss, total soluble solids, total sugar, TSS/Acid ratio, bitter pit incidence and soft rot as compared to control. A significant increase in juice content, starch score, titratable acidity, ascorbic acid, fruit flesh firmness and fruit density were recorded with CaCl2 application.

Conclusions

o CaCl2 application has significantly retained high juice content, ascorbic acid, fruit flesh firmness and least bitter pit incidence in cultivar Royal Gala. o Apple cultivar Mondial Gala is characterized by least weight loss, high titratable acidity and relatively low bitter pit incidence, thus can be stored for long term storage. o Cultivar Golden Delicious has poor quality as well as the least fruit flesh firmness and the highest bitter pit incidence, thus can not be recommended for prolong storage.

o CaCl2 application has not only improved the quality of apple fruit but also decreased the incidence of bitter pit and soft rot.

Recommendations

 CaCl2 application has enhanced the quality of cultivar Royal Gala by retaining high juice content, ascorbic acid, fruit flesh firmness and least bitter pit incidence during storage in cultivar Royal Gala, thus can be recommended for extended storage.  Apple cultivar Mondial Gala is characterized by least weight loss, high

titratable acidity and relatively low bitter pit incidence with CaCl2 application, thus measures must be taken to further decrease the bitter pit incidence during storage.

 CaCl2 application has not only improved the quality of apple fruit but also

decreased the incidence of bitter pit and soft rot, therefore CaCl2 application is strongly recommended for better storage performance of apple fruits.

BACKGROUND This postharvest study was carried out on a range of apple cultivars, storage duration and calcium concentration and dipping duration. The first experiment of this study was focused to quantify the storage duration for apple cultivars. Six storage durations (0, 30, 60, 90, 120 and 150 days) were used. In the second experiment storage durations were restricted to 150 days; however, Royal Gala, Mondial Gala, Golden Delicious and Red Delicious were used to identify the best cultivar for storage. In the second experiment, these cultivars were evaluated at different times of maturity. In this study Red Delicious cultivar was superior to other cultivars. Therefore, Red Delicious cultivar was further investigated for only two storage durations, four calcium dipping and four calcium durations. This study concluded that calcium chloride dipped of 9% and duration of 12 minutes was the best treatment. Later on in experiment 4, it was decided to include all cultivars and test them with selected storage duration and calcium treatments. Based on the entire research program the following conclusions and recommendations can be drawn. OVERALL CONCLUSIONS AND RECOMMENDATIONS

The data recorded during the research on “Influence of storage duration, harvesting stage and CaCl2 treatment on storage performance of apple cultivars” revealed that:

 The apple cultivar Red Delicious has the highest quality attributes but was found to be most susceptible to bitter and soft rot incidence, especially with prolong storage, thus measures such as pre and post harvest calcium application should be a regular exercise to minimize the better pit and soft rot incidence during storage.  Cultivar Golden Delicious has the lowest ascorbic acid and highest weight loss, thus measures such as optimizing relative humidity should be taken to decrease the rate of weight loss during long term storage of this cultivar.  Cultivar Mondial Gala being the lowest in fruit flesh firmness, ascorbic acid, starch content is relatively poor in quality but has the least soft rot incidence, while cultivar Royal Gala has the lowest incidence of bitter pit but has poor quality characteristics. Thus, both cultivars can be stored for longer duration than Golden Delicious or Red Delicious.  Early harvested fruits, despite low incidence of soft rot, were found to have the highest weight loss, bitter pit as well as the least juice content, TSS, total sugar and TSS/Acid ratio. By contrast, fruits harvested at mid harvesting stage having relatively good quality and storage performance as compared to both early and late harvested fruits. Therefore, the practice of both early and late harvesting should be replaced with harvesting at the optimum maturity stage (Mid harvesting stage) to ensure both quality and longer storage life.

 Fruits dipped in 9% CaCl2 solution for 12 minutes retained most of the quality attributes and decreased the bitter pit incidence. Therefore, the same practise is recommended for the commercial storage of apple fruits.

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APPENDICES

Appendix 1. ANOVA for weight loss (%) of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 4.65 1.55 9.25 0.00 Storage durations (S) 5 187.29 37.46 223.28 0.00 C × S 15 2.84 0.19 1.13 0.36 Error 48 8.05 0.17 Total 71 202.83

Appendix 2. ANOVA for juice content (%) of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 602.64 200.88 23.24 0.00 Storage durations (S) 5 3788.02 757.60 87.67 0.00 C × S 15 116.60 7.77 0.90 Error 48 414.81 8.64 Total 71 4922.07

Appendix 3. ANOVA for starch content (Score) of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 3.79 1.26 5.38 0.00 Storage durations (S) 5 186.43 37.29 158.86 0.00 C × S 15 7.59 0.51 2.16 0.02 Error 48 11.27 0.24 Total 71 209.07

Appendix 4. ANOVA for total soluble solids of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 0.44 0.15 0.45 Storage durations (S) 5 60.24 12.05 36.92 0.00 C × S 15 2.84 0.19 0.58 Error 48 15.67 0.33 Total 71 79.18

Appendix 5. ANOVA for total sugar (%) of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 4.04 1.35 3.15 0.03 Storage durations (S) 5 88.08 17.62 41.17 0.00 C × S 15 2.32 0.16 0.36 Error 48 20.54 0.43 Total 71 114.98

Appendix 6. ANOVA for titratable acidity (%) of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 0.06 0.02 10.51 0.00 Storage durations (S) 5 0.80 0.16 88.64 0.00 C × S 15 0.01 0.001 0.47 Error 48 0.09 0.002 Total 71 0.95

Appendix 7. ANOVA for pH of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 0.28 0.09 0.90 Storage durations (S) 5 4.87 0.97 9.40 0.00 C × S 15 0.49 0.03 0.32 Error 48 4.97 0.10 Total 71 10.60

Appendix 8. ANOVA for TSS/Acid ratio of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 223.74 74.58 11.41 0.00 Storage durations (S) 5 3684.47 736.89 112.77 0.00 C × S 15 59.23 3.95 0.60 Error 48 313.66 6.54 Total 71 4281.09

Appendix 9. ANOVA for ascorbic acid (mg/100g) of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 100.14 33.38 75.63 0.00 Storage durations (S) 5 223.65 44.73 101.35 0.00 C × S 15 22.24 1.48 3.36 0.00 Error 48 21.19 0.44 Total 71 367.21

Appendix 10. ANOVA for fruit flesh firmness (kg/cm2) of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 8.35 2.78 48.68 0.00 Storage durations (S) 5 61.21 12.24 214.08 0.00 C × S 15 0.76 0.05 0.88 Error 48 2.75 0.06 Total 71 73.06

Appendix 11. ANOVA for fruit density (g/cm3) of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 0.02 0.007 4.46 0.00 Storage durations (S) 5 0.02 0.004 2.48 0.04 C × S 15 0.001 0.0001 0.04 Error 48 0.08 0.002 Total 71 0.12

Appendix 12. ANOVA for bitter pit (%) of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 539.71 179.90 197.59 0.00 Storage durations (S) 5 3054.37 610.87 670.93 0.00 C × S 15 547.68 36.51 40.10 0.00 Error 48 43.70 0.91 Total 71 4185.46

Appendix 13. ANOVA for soft rot (%) of apple cultivars as affected by storage durations

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 259.88 86.63 33.69 0.00 Storage durations (S) 5 5120.80 1024.16 398.29 0.00 C × S 15 155.69 10.38 4.04 0.00 Error 48 123.43 2.57 Total 71 5659.80

Appendix 14. ANOVA for weight loss (%) of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 2.61 0.87 10.84 0.00 Storage durations (S) 1 452.86 452.86 5639.53 0.00 C × S 3 2.61 0.87 10.84 0.00 Harvesting stages (H) 2 25.98 12.99 161.79 0.00 C × H 6 1.24 0.21 2.57 0.03 S × H 2 25.98 12.99 161.79 0.00 C × S × H 6 1.24 0.21 2.57 0.03 Error 48 3.85 0.08 Total 71 516.37

Appendix 15. ANOVA for juice content (%) of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 477.81 159.27 6.29 0.00 Storage durations (S) 1 3448.24 3448.24 136.09 0.00 C × S 3 7.35 2.45 0.10 Harvesting stages (H) 2 1730.91 865.45 34.16 0.00 C × H 6 23.60 3.93 0.16 S × H 2 6.71 3.36 0.13 C × S × H 6 10.85 1.81 0.07 Error 48 1216.20 25.34 Total 71 6921.66

Appendix 16. ANOVA for starch content (score) of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 2.15 0.72 1.74 0.17 Storage durations (S) 1 354.58 354.58 863.35 0.00 C × S 3 0.37 0.12 0.30 Harvesting stages (H) 2 38.42 19.21 46.77 0.00 C × H 6 3.70 0.62 1.50 0.20 S × H 2 0.002 0.001 0.002 C × S × H 6 1.21 0.20 0.49 Error 48 19.71 0.41 Total 71 420.13

Appendix 17. ANOVA for total soluble solids of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 4.24 1.41 4.77 0.01 Storage durations (S) 1 154.53 154.53 521.75 0.00 C × S 3 0.11 0.04 0.12 Harvesting stages (H) 2 97.39 48.70 164.42 0.00 C × H 6 0.09 0.02 0.05 S × H 2 6.28 3.14 10.61 0.00 C × S × H 6 0.39 0.07 0.22 Error 48 14.22 0.30 Total 71 277.25

Appendix 18. ANOVA for total sugar (%) of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 3.49 1.16 3.54 0.02 Storage durations (S) 1 139.78 139.78 425.93 0.00 C × S 3 0.52 0.18 0.53 Harvesting stages (H) 2 163.52 81.76 249.13 0.00 C × H 6 1.53 0.26 0.78 S × H 2 4.75 2.38 7.24 0.00 C × S × H 6 3.51 0.58 1.78 0.12 Error 48 15.75 0.33 Total 71 332.85

Appendix 19. ANOVA for titratable acidity (%) of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 0.07 0.02 14.02 0.00 Storage durations (S) 1 1.83 1.83 1105.70 0.00 C × S 3 0.004 0.001 0.83 Harvesting stages (H) 2 0.14 0.07 42.22 0.00 C × H 6 0.005 0.001 0.54 S × H 2 0.04 0.02 12.04 0.00 C × S × H 6 0.003 0.001 0.35 Error 48 0.08 0.002 Total 71 2.18

Appendix 20. ANOVA for pH of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 0.21 0.07 0.79 Storage durations (S) 1 11.88 11.88 131.01 0.00 C × S 3 0.94 0.31 3.45 0.02 Harvesting stages (H) 2 3.33 1.67 18.37 0.00 C × H 6 0.08 0.01 0.15 S × H 2 0.27 0.13 1.48 0.24 C × S × H 6 0.12 0.02 0.23 Error 48 4.35 0.09 Total 71 21.19

Appendix 21. ANOVA for TSS/Acid ratio of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 208.27 69.42 7.25 0.00 Storage durations (S) 1 3803.66 3803.66 396.95 0.00 C × S 3 62.05 20.68 2.16 0.11 Harvesting stages (H) 2 1341.25 670.62 69.99 0.00 C × H 6 8.63 1.44 0.15 S × H 2 48.57 24.28 2.53 0.09 C × S × H 6 23.39 3.90 0.41 Error 48 459.95 9.58 Total 71 5955.78

Appendix 22. ANOVA for ascorbic acid (mg/100g) of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 44.86 14.95 40.88 0.00 Storage durations (S) 1 545.60 545.60 1491.40 0.00 C × S 3 11.53 3.84 10.51 0.00 Harvesting stages (H) 2 70.56 35.28 96.44 0.00 C × H 6 1.51 0.25 0.69 S × H 2 2.72 1.36 3.71 0.03 C × S × H 6 3.22 0.54 1.47 0.21 Error 48 17.56 0.37 Total 71 697.57

Appendix 23. ANOVA for fruit flesh firmness (kg/cm2) of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 7.01 2.34 20.46 0.00 Storage durations (S) 1 112.80 112.80 988.07 0.00 C × S 3 0.58 0.19 1.68 0.18 Harvesting stages (H) 2 13.55 6.78 59.35 0.00 C × H 6 0.09 0.02 0.13 S × H 2 0.03 0.01 0.11 C × S × H 6 0.09 0.02 0.14 Error 48 5.48 0.11 Total 71 139.62

Appendix 24. ANOVA for fruit density (g/cm3) of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 0.02 0.01 10.34 0.00 Storage durations (S) 1 0.05 0.05 66.27 0.00 C × S 3 0.00 0.00 0.17 Harvesting stages (H) 2 0.08 0.04 55.05 0.00 C × H 6 0.00 0.00 0.66 S × H 2 0.00 0.00 0.90 C × S × H 6 0.00 0.00 0.31 Error 48 0.03 0.00 Total 71 0.19

Appendix 25. ANOVA for soft rot (%) of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 284.72 94.91 21.84 0.00 Storage durations (S) 1 11085.61 11085.61 2551.27 0.00 C × S 3 284.72 94.91 21.84 0.00 Harvesting stages (H) 2 389.42 194.71 44.81 0.00 C × H 6 18.97 3.16 0.73 S × H 2 389.42 194.71 44.81 0.00 C × S × H 6 18.97 3.16 0.73 Error 48 208.57 4.35 Total 71 12680.41

Appendix 26. ANOVA for bitter pit (%) of apple cultivars as affected by storage and harvesting stages

SOV DF SS MS F-Cal P-Value Cultivars (C) 3 957.87 319.29 67.27 0.00 Storage durations (S) 1 6141.64 6141.64 1293.95 0.00 C × S 3 957.87 319.29 67.27 0.00 Harvesting stages (H) 2 307.46 153.73 32.39 0.00 C × H 6 41.68 6.95 1.46 0.21 S × H 2 307.46 153.73 32.39 0.00 C × S × H 6 41.68 6.95 1.46 0.21 Error 48 227.83 4.75 Total 71 8983.49

Appendix 27. ANOVA for weight loss (%) of apple fruit as affected by storage, CaCl2 concentrations and dipping durations

SOV DF SS MS F-Cal P-Value Storage (S) 1 252.14 252.14 15923.38 0.00 Dipping durations (D) 3 2.08 0.69 43.71 0.00 S × D 3 2.08 0.69 43.71 0.00 CaCl2 conc. (Ca) 3 5.25 1.75 110.52 0.00 S × Ca 3 5.25 1.75 110.52 0.00 D × Ca 9 0.65 0.07 4.52 0.00 S × D × Ca 9 0.65 0.07 4.52 0.00 Error 64 1.01 0.02 Total 95 269.09

Appendix 28. ANOVA for weight loss (%) of apple fruit as affected by storage, CaCl2 concentrations and dipping durations

SOV DF SS MS F-Cal P-Value Storage (S) 1 4940.71 4940.71 620.12 0.00 Dipping durations (D) 3 16.25 5.42 0.68 S × D 3 9.70 3.24 0.41 CaCl2 conc. (Ca) 3 182.53 60.85 7.64 0.00 S × Ca 3 121.21 40.41 5.07 0.00 D × Ca 9 4.35 0.48 0.06 S × D × Ca 9 5.02 0.56 0.07 Error 64 509.91 7.97 Total 95 5789.70

Appendix 29. ANOVA for starch content (score) of apple fruit as affected by storage, CaCl2 concentrations and dipping durations

SOV DF SS MS F-Cal P-Value Storage (S) 1 274.96 274.96 36414.02 0.00 Dipping durations (D) 3 0.60 0.20 26.29 0.00 S × D 3 0.26 0.09 11.24 0.00 CaCl2 conc. (Ca) 3 5.47 1.82 241.24 0.00 S × Ca 3 1.03 0.34 45.35 0.00 D × Ca 9 0.26 0.03 3.78 0.00 S × D × Ca 9 0.13 0.01 1.86 0.07 Error 64 0.48 0.01 Total 95 283.17

Appendix 30. ANOVA for total soluble solids of apple fruit as affected by storage, CaCl2 concentrations and dipping durations

SOV DF SS MS F-Cal P-Value Storage (S) 1 124.94 124.94 2100.12 0.00 Dipping durations (D) 3 0.50 0.17 2.78 0.05 S × D 3 0.53 0.18 2.99 0.04 CaCl2 conc. (Ca) 3 0.76 0.25 4.26 0.01 S × Ca 3 1.06 0.35 5.94 0.00 D × Ca 9 0.23 0.03 0.43 S × D × Ca 9 0.26 0.03 0.48 Error 64 3.81 0.06 Total 95 132.08

Appendix 31. ANOVA for total sugar (%) of apple fruit as affected by storage, CaCl2 concentrations and dipping durations

SOV DF SS MS F-Cal P-Value Storage (S) 1 182.93 182.93 5851.12 0.00 Dipping durations (D) 3 0.20 0.07 2.18 0.10 S × D 3 0.62 0.21 6.57 0.00 CaCl2 conc. (Ca) 3 0.48 0.16 5.10 0.00 S × Ca 3 1.66 0.55 17.71 0.00 D × Ca 9 0.09 0.01 0.30 S × D × Ca 9 0.23 0.03 0.82 Error 64 2.00 0.03 Total 95 188.21

Appendix 32. ANOVA for titratable acidity (%) of apple cultivars as affected by storage, CaCl2 concentrations and dipping durations

SOV DF SS MS F-Cal P-Value Storage (S) 1 0.67 0.67 354.88 0.00 Dipping durations (D) 3 0.00 0.00 0.48 S × D 3 0.03 0.01 4.98 0.00 CaCl2 conc. (Ca) 3 0.02 0.01 3.62 0.02 S × Ca 3 0.08 0.03 13.35 0.00 D × Ca 9 0.00 0.00 0.23 S × D × Ca 9 0.01 0.00 0.76 Error 64 0.12 0.00 Total 95 0.94

Appendix 33. ANOVA for TSS/Acid ratio of apple fruit as affected by storage, CaCl2 concentrations and dipping durations

SOV DF SS MS F-Cal P-Value Storage (S) 1 5449.76 5449.76 1317.18 0.00 Dipping durations (D) 3 52.42 17.48 4.22 0.01 S × D 3 165.27 55.09 13.31 0.00 CaCl2 conc. (Ca) 3 277.38 92.46 22.35 0.00 S × Ca 3 482.52 160.84 38.87 0.00 D × Ca 9 41.89 4.66 1.13 0.36 S × D × Ca 9 78.58 8.73 2.11 0.04 Error 64 264.80 4.14 Total 95 6812.63

Appendix 34. ANOVA for ascorbic acid (mg/100g) of apple fruit as affected by storage, CaCl2 concentrations and dipping durations

SOV DF SS MS F-Cal P-Value Storage (S) 1 304.81 304.81 564.89 0.00 Dipping durations (D) 3 2.70 0.90 1.66 0.18 S × D 3 2.66 0.89 1.64 0.19 CaCl2 conc. (Ca) 3 19.26 6.42 11.90 0.00 S × Ca 3 15.75 5.25 9.73 0.00 D × Ca 9 0.89 0.10 0.18 S × D × Ca 9 0.86 0.10 0.18 Error 64 34.53 0.54 Total 95 381.45

Appendix 35. ANOVA for fruit flesh firmness (kg/cm2) of apple fruit as affected by storage, CaCl2 concentrations and dipping durations

SOV DF SS MS F-Cal P-Value Storage (S) 1 99.02 99.02 2043.43 0.00 Dipping durations (D) 3 1.96 0.65 13.50 0.00 S × D 3 0.91 0.30 6.28 0.00 CaCl2 conc. (Ca) 3 4.24 1.41 29.13 0.00 S × Ca 3 1.03 0.34 7.11 0.00 D × Ca 9 0.54 0.06 1.23 0.29 S × D × Ca 9 0.46 0.05 1.06 0.40 Error 64 3.10 0.05 Total 95 111.27

Appendix 36. ANOVA for fruit density (g/cm3) of apple fruit as affected by storage, CaCl2 concentrations and dipping durations

SOV DF SS MS F-Cal P-Value Storage (S) 1 0.059 0.059 693.444 0.000 Dipping durations (D) 3 0.005 0.002 20.259 0.000 S × D 3 0.000 0.000 0.704 CaCl2 conc. (Ca) 3 0.008 0.003 30.037 0.000 S × Ca 3 0.008 0.003 30.037 0.000 D × Ca 9 0.000 0.000 0.210 S × D × Ca 9 0.000 0.000 0.210 Error 64 0.005 0.000 Total 95 0.085

Appendix 37. ANOVA for soft rot (%) of apple fruit as affected by storage, CaCl2 concentrations and dipping durations

SOV DF SS MS F-Cal P-Value Storage (S) 1 4338.03 4338.03 18451.85 0.00 Dipping durations (D) 3 9.21 3.07 13.06 0.00 S × D 3 9.21 3.07 13.06 0.00 CaCl2 conc. (Ca) 3 2561.24 853.75 3631.41 0.00 S × Ca 3 2561.24 853.75 3631.41 0.00 D × Ca 9 3.84 0.43 1.82 0.08 S × D × Ca 9 3.84 0.43 1.82 0.08 Error 64 15.05 0.24 Total 95 9501.66

Appendix 38. ANOVA for bitter pit (%) of apple fruit as affected by storage, CaCl2 concentrations and dipping durations

SOV DF SS MS F-Cal P-Value Storage (S) 1 6897.97 6897.97 909.76 0.00 Dipping durations (D) 3 547.55 182.52 24.07 0.00 S × D 3 547.55 182.52 24.07 0.00 CaCl2 conc. (Ca) 3 1746.61 582.20 76.79 0.00 S × Ca 3 1746.61 582.20 76.79 0.00 D × Ca 9 211.33 23.48 3.10 0.00 S × D × Ca 9 211.33 23.48 3.10 0.00 Error 64 485.26 7.58 Total 95 12394.22

Appendix 39. ANOVA for weight loss (%) of apple cultivars as affected by storage and CaCl2

SOV DF SS MS F-Cal P-Value Cultivars (C) 2 2.29 1.15 32.85 0.00 Storage durations (S) 1 111.20 111.20 3189.97 0.00 C × S 2 2.29 1.15 32.85 0.00 CaCl2 conc. (Ca) 1 12.92 12.92 370.76 0.00 C × Ca 2 0.08 0.04 1.20 0.32 S × Ca 1 12.92 12.92 370.76 0.00 C × S × Ca 2 0.08 0.04 1.20 0.32 Error 24 0.84 0.04 Total 35 142.63

Appendix 40. ANOVA for juice content (%) of apple cultivars as affected by storage and CaCl2

SOV DF SS MS F-Cal P-Value Cultivars (C) 2 700.15 350.07 12.23 0.00 Storage durations (S) 1 2365.20 2365.20 82.64 0.00 C × S 2 37.80 18.90 0.66 CaCl2 conc. (Ca) 1 180.45 180.45 6.30 0.02 C × Ca 2 52.81 26.40 0.92 S × Ca 1 310.93 310.93 10.86 0.00 C × S × Ca 2 131.85 65.92 2.30 0.12 Error 24 686.93 28.62 Total 35 4466.13

Appendix 41. ANOVA for starch content (score) of apple cultivars as affected by storage and CaCl2

SOV DF SS MS F-Cal P-Value Cultivars (C) 2 0.27 0.14 1.40 0.26 Storage durations (S) 1 124.66 124.66 1295.40 0.00 C × S 2 0.11 0.05 0.56 CaCl2 conc. (Ca) 1 5.67 5.67 58.95 0.00 C × Ca 2 0.67 0.34 3.49 0.05 S × Ca 1 7.19 7.19 74.73 0.00 C × S × Ca 2 0.76 0.38 3.95 0.03 Error 24 2.31 0.10 Total 35 141.64

Appendix 42. ANOVA for total soluble solids (0Brix) of apple cultivars as affected by storage and CaCl2

SOV DF SS MS F-Cal P-Value Cultivars (C) 2 0.42 0.21 1.71 0.20 Storage durations (S) 1 58.22 58.22 474.19 0.00 C × S 2 0.07 0.04 0.30 CaCl2 conc. (Ca) 1 1.26 1.26 10.28 0.00 C × Ca 2 0.05 0.02 0.18 S × Ca 1 1.66 1.66 13.55 0.00 C × S × Ca 2 0.06 0.03 0.22 Error 24 2.95 0.12 Total 35 64.68

Appendix 43. ANOVA for total sugar (%) of apple cultivars as affected by storage and CaCl2

SOV DF SS MS F-Cal P-Value Cultivars (C) 2 1.32 0.66 3.28 0.06 Storage durations (S) 1 58.65 58.65 292.36 0.00 C × S 2 0.39 0.20 0.98 CaCl2 conc. (Ca) 1 2.49 2.49 12.42 0.00 C × Ca 2 0.03 0.02 0.08 S × Ca 1 2.90 2.90 14.43 0.00 C × S × Ca 2 0.03 0.02 0.08 Error 24 4.82 0.20 Total 35 70.62

Appendix 44. ANOVA for titratable acidity (%) of apple cultivars as affected by storage and CaCl2

SOV DF SS MS F-Cal P-Value Cultivars (C) 2 0.03 0.01 11.75 0.00 Storage durations (S) 1 0.55 0.55 472.61 0.00 C × S 2 0.03 0.01 12.04 0.00 CaCl2 conc. (Ca) 1 0.02 0.02 12.72 0.00 C × Ca 2 0.00 0.00 0.07 S × Ca 1 0.02 0.02 18.06 0.00 C × S × Ca 2 0.00 0.00 0.03 Error 24 0.03 0.00 Total 35 0.67

Appendix 45. ANOVA for TSS/Acid ratio of apple cultivars as affected by storage and CaCl2

SOV DF SS MS F-Cal P-Value Cultivars (C) 2 41.24 20.62 4.42 0.02 Storage durations (S) 1 2563.06 2563.06 549.08 0.00 C × S 2 29.79 14.89 3.19 0.06 CaCl2 conc. (Ca) 1 224.60 224.60 48.12 0.00 C × Ca 2 0.22 0.11 0.02 S × Ca 1 246.91 246.91 52.90 0.00 C × S × Ca 2 0.10 0.05 0.01 Error 24 112.03 4.67 Total 35 3217.95

Appendix 46. ANOVA for ascorbic acid (mg/100g) of apple cultivars as affected by storage and CaCl2

SOV DF SS MS F-Cal P-Value Cultivars (C) 2 32.69 16.34 41.40 0.00 Storage durations (S) 1 147.14 147.14 372.66 0.00 C × S 2 4.68 2.34 5.93 0.01 CaCl2 conc. (Ca) 1 24.90 24.90 63.06 0.00 C × Ca 2 0.18 0.09 0.23 S × Ca 1 19.10 19.10 48.37 0.00 C × S × Ca 2 0.16 0.08 0.20 Error 24 9.48 0.40 Total 35 238.32

Appendix 47. ANOVA for fruit flesh firmness (kg/cm2) of apple cultivars as affected by storage and CaCl2

SOV DF SS MS F-Cal P-Value Cultivars (C) 2 2.67 1.34 22.46 0.00 Storage durations (S) 1 42.23 42.23 709.72 0.00 C × S 2 0.58 0.29 4.89 0.02 CaCl2 conc. (Ca) 1 4.88 4.88 81.96 0.00 C × Ca 2 0.08 0.04 0.71 S × Ca 1 2.30 2.30 38.58 0.00 C × S × Ca 2 0.05 0.02 0.41 Error 24 1.43 0.06 Total 35 54.22

Appendix 48. ANOVA for fruit density (g/cm3) of apple cultivars as affected by storage and CaCl2

SOV DF SS MS F-Cal P-Value Cultivars (C) 2 0.02 0.01 16.16 0.00 Storage durations (S) 1 0.03 0.03 39.20 0.00 C × S 2 0.00 0.00 3.11 0.06 CaCl2 conc. (Ca) 1 0.00 0.00 5.48 0.03 C × Ca 2 0.00 0.00 0.68 S × Ca 1 0.00 0.00 2.50 0.13 C × S × Ca 2 0.00 0.00 0.60 Error 24 0.02 0.00 Total 35 0.08

Appendix 49. ANOVA for bitter pit (%) of apple cultivars as affected by storage and CaCl2

SOV DF SS MS F-Cal P-Value Cultivars (C) 2 118.66 59.33 143.52 0.00 Storage durations (S) 1 698.90 698.90 1690.60 0.00 C × S 2 118.66 59.33 143.52 0.00 CaCl2 conc. (Ca) 1 410.33 410.33 992.57 0.00 C × Ca 2 56.58 28.29 68.43 0.00 S × Ca 1 410.33 410.33 992.57 0.00 C × S × Ca 2 56.58 28.29 68.43 0.00 Error 24 9.92 0.41 Total 35 1879.96

Appendix 50. ANOVA for soft rot (%) of apple cultivars as affected by storage and CaCl2

SOV DF SS MS F-Cal P-Value Cultivars (C) 2 17.49 8.75 2.09 0.15 Storage durations (S) 1 1005.42 1005.42 240.15 0.00 C × S 2 17.49 8.75 2.09 0.15 CaCl2 conc. (Ca) 1 540.95 540.95 129.21 0.00 C × Ca 2 8.28 4.14 0.99 S × Ca 1 540.95 540.95 129.21 0.00 C × S × Ca 2 8.28 4.14 0.99 Error 24 100.48 4.19 Total 35 2239.35